We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread  Spread of infection to new hosts requires reactivation of the latent infection and subsequent transport of progeny virions in axons to distant egress sites in the periphery Enveloped viral particles transport within the lumen of vesicles, likely derived from the trans-Golgi network (TGN) The subcellular localization of Us9 is critical for protein functionality. Us9 is enriched in lipid raft membrane microdomains, localization to which is essential for Us9-mediated anterograde transport of virions in vivo. Two tyrosine (Y49 and Y50) residues within the cluster, known not to be phosphorylated or influence subcellular localization, are essential for productive anterograde spread in vivoThough no crystal structure has been established for Us9, one critical domain is a 10-amino acid cluster of negatively charged acidic residues (46–55) in vivoin vitroTwo serine residues (S51 and S53) within the acidic cluster are phosphorylated, as determined through radiolabelling assays in vitro at the cellular and biochemical level. We visualized the distribution of phosphorylated Us9 in axons using immunofluorescence with a phospho-specific antibody and then analyzed the partitioning of phosphorylated and total Us9 protein species across different membrane microdomains. For live cell imaging and for quantification of anterograde spread, we isolated PRV recombinants expressing GFP-tagged, mutant Us9 variants with alanine substituted for one or both serine residues. We then performed infections of chambered neuronal cultures to quantify the spread defect associated with loss of phosphorylation. Finally, we assessed Us9-Kif1A interactions in the serine mutant background through co-immunoprecipitation experiments. This study of the role of Us9 phosphorylation in anterograde sorting, transport, and spread expands our understanding of the Us9 functional domains.In this study, we characterized serine residues 51 and 53 of the PRV Us9 protein We employed the wild type PRV Becker and derivative strains PRV 171 and PRV 173 expressing Us9 acidic cluster point mutants For this study, we constructed three new PRV strains  expressing GFP-tagged mutant Us9 proteins. Site-directed mutagenesis of the GFP-Us9 N-terminal fusion plasmid pML122, originally used to construct PRV 340, was performed to introduce missense mutations  at S51, S53, or both. Three sets of primers (forward and reverse) with the following nucleotide sequences were employed, (mutagenic codon underlined):S51A:GCCGAGAGCGACAACGAGACG 3′5′ GACTCGGACTGCTACTACF: GGCGTAGTAGCAGTCCGAGTC 3′5′ CGTCTCGTTGTCGCTCTCR: S53A:GCCGACAACGAGACGCCCAGC 3′5′ GACTGCTACTACAGCGAGF: CGGCCTCGCTGTAGTAGCAGTC 3′5′ GCTGGGCGTCTCGTTGTR: S51,53A:GCCGAGGCCGACAACGAGACG 3′5′ GACTCGGACTGCTACTACF: GGCCTCGGCGTAGTAGCAGTCCGAGTC 3′5′ CGTCTCGTTGTCR: Us4 genome locus 7 cells per 10cm dish were transfected with PRV 337 nucleocapsid DNA and 4ug NsiI-digested pRK05, pRK06, or pRK07 respectively using Lipofectamine 2000, following the manufacturer's instructions . After significant cytopathic effect (CPE) developed (∼48 hours post-transfection), the cells were harvested and the media plated onto PK15 cells at serial dilutions to isolate individual recombinant plaques expressing the GFP fusion protein. Final viral stocks were assessed by western blot (WB) for expression of the GFP-Us9 fusion and loss of BHV Us9 expression. Three strains were isolated: PRV 451 , PRV 452 (GFP-Us9 S51A), and PRV 453 (GFP-Us9 S53A).Site-directed mutagenesis using the QuikChange II XL kit, performed according to the manufacturer's instructions , yielded the plasmids pRK05 , pRK06 (GFP-Us9 S51A), and pRK07 (GFP-Us9 S53A). Each plasmid was sequenced to confirm mutagenesis and then recombined into the PRV genome through co-transfection with purified nucleocapsid DNA of PRV 337 Dual fluorescent strains expressing both serine mutant GFP-Us9 and the mRFP-Vp26 capsid fusion were constructed. PK15 cells were coinfected with PRV 451, PRV 452, or PRV 453 and PRV 325  include rabbit polyclonal anti-Us9 serum , 1∶1000,A novel phospho-specific Us9 mouse monoclonal antibody was isolated by Genscript  for this study. Briefly, BALB/c mice were immunized with a KLH-conjugated peptide mimicking the Us9 acidic cluster sequence, which contains the two putative serine phosphorylation sites rd day for 11 days before infection. At this time, fully differentiated cells have exited the cell cycle and have developed neurites which exhibit axonal polarity, i.e. stain positive for the phosphorylated neurofilament H and Tau markers Porcine kidney 15 (PK15) cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The transformed neuronal cell line PC12 Dissociated cultures of primary rat superior cervical ganglia (SCG) neurons were prepared as previously detailed 5 cells in neuronal media supplemented with 1% FBS) was plated in the N compartment 24 hours before any infections to amplify virus spread into this compartment.Campenot chambered cultures of SCG neurons have been employed extensively in our laboratory to assay directional spread of PRV Infections of PK15, PC12, dissociated and chambered SCG cultures were performed as previously described Isolation of detergent-resistant membrane (DRM) fractions, or lipid rafts, was performed through an OptiPrep sucrose gradient  To isolate and assess Us9 binding partners, we employed a recently described method that utilizes high affinity anti-GFP antibodies as probes for co-immunoprecipitation of viral protein complexes Infected cell samples to be analyzed by WB without lipid raft flotation or co-immunoprecipitation were lysed with RSB, 1% NP40 detergent. Lysis conditions for lipid raft float or co-immunoprecipitation samples have been described above. All samples were then heated to 65C for 10 minutes, mixed 1∶6 with 6X Laemmli sample buffer, run on 12% 1-D SDS-PAGE gels, and transferred to a PVDF membrane. Antibodies and dilution factors employed are described above. Primary antibody was diluted in 5% milk in tris-buffered saline, supplemented with 0.1% tween (TBS-T), and applied to the membrane for 1 hour at room temperature. Membranes were then washed 3 times with TBS-T, and secondary HRP-conjugated antibody was applied for 1 hour at room temperature. After 3 subsequent washes with TBS-T, signal was detected using the chemiluminescent kit according to the manufacturer's instructors (Thermo-Fischer Scientific). For experiments requiring quantitation of WB signals, each protein sample was subject to a two-fold dilution series and the chemiluminescent signals were detected at each dilution to cover the linear and non-linear range of detection for the primary antibody.Phosphorylated Us9 subspecies were visualized in axons of PRV-infected dissociated SCG cultures through immunofluorescence (IF). Cultures were infected with PRV 340 and fixed at 8 hours post-infection with 4% paraformaldehyde. Samples were washed 3 times with PBS and then blocked with 3% bovine serum album (BSA) in PBS for at least 1 hour before permeablization with 0.5% saponin in 3% BSA/PBS for 30 minutes. Primary antibody  was diluted in the same permeablization solution and applied for 1 hour, followed by 3 washes with PBS. Secondary antibody (goat anti-mouse-IgG conjugated with AlexaFluor 568) was diluted in permeablization solution and applied for 1 hour, followed by 3 washes with PBS. Samples were then visualized with a Nikon Ti-Eclipse epifluorescent inverted microscope. As a negative control to establish non-specific antibody binding and background fluorescence, cultures were infected with PRV 451 encoding Us9 not detectable by 2D5E6 and analyzed under the same IF conditions.Live cell imaging of the recombinant fluorescent strains PRV 341, PRV 454, PRV 455, and PRV 456 was performed in dissociated SCG cultures as previously described for the wild-type GFP-Us9 fusion protein We first utilized immunofluorescence microscopy to analyze the subcellular localization of phosphorylated Us9 subspecies (phospho-Us9) with the newly derived monoclonal phospho-specific antibody 2D5E6 . At 8 hoPrevious studies have demonstrated that Us9 is enriched in lipid raft microdomains and that this localization pattern is essential for anterograde transport Phospho-Us9 is readily detectable in both raft and non-raft membranes, and it is unclear if this distribution reflects two different subpopulations or an equilibrium between association of the protein with different membrane microenvironments. To assess whether phosphorylation was dependent on association with a lipid raft, we infected differentiated PC12 cultures with PRV 322, expressing Us9 targeted to non-raft domains, and performed lipid raft isolation and WB analysis for phosphorylation as above. As previously described for PRV 322 infection in vivo work where PRV 173 was found to be defective for anterograde spread when compared to wild-type PRV Becker at equivalent timepoints but could undergo some spread at later times during infection in vivo and is not efficiently phosphorylated To assess the functional relevance of phosphorylation to Us9-mediated anterograde transport of PRV, we infected the cell bodies of chambered SCG neuron cultures with a panel of recombinant strains expressing different Us9 variants. We assayed the anterograde spread capacity at 24 hours post-infection for PRV strains expressing wild type Us9 (PRV Becker or PRV 340) as well as several mutant Us9 variants  . AnterogWe then compared spread of infection for three GFP-tagged Us9 serine mutants to wild-type GFP-Us9. Loss of only one serine residue (51 or 53) had no detectable defect on anterograde spread (PRV 452 or 453). However, loss of both serine phosphorylation sites resulted in a ∼1.5log defect compared to wild-type GFP-Us9. Overall, we find that serine phosphorylation is not absolutely required for anterograde spread, though it may affect the kinetic efficiency of this process.To further characterize the importance of Us9 phosphorylation, we visualized virion transport during infection with several Us9 mutants. Dissociated SCG neurons were infected with PRV 341, 454, 455, or 456, and cells were imaged at 8 hours post-infection. No difference in the localization of Us9 was observed in cell bodies for these strains, with robust GFP-Us9 signal detected in the plasma membrane, as well as on intracellular vesicles (data not shown). Anterograde transport of mature virions, i.e. GFP-Us9 (+)/mRFP-Vp26 (+) punctate structures, was observed for PRV 454, 455, and 456 , S2, S3,As anterograde transport of virions was reduced during infection with diserine mutant Us9, we next determined if phosphorylation affects Us9-Kif1A binding, a key step in the axonal targeting of viral particles in vivo studies of mutant viral strains. In this study, we characterized the role of Us9 phosphorylation at serines 51 and 53 in anterograde transport using biochemical fractionation and cellular imaging techniques that complement and expand upon the previously described spread deficiency attributed to these residues in vivoUnderstanding the molecular mechanisms underlying axonal sorting and subsequent anterograde transport of alphaherpes viruses is necessary for a rigorous characterization of viral spread and can complement in vitro. Our previous model held that these sites formed the CK2 consensus sequence and were therefore necessary for phosphorylation of serines 51 and 53. However, since a single mutation of serine 51 or 53 has no effect on spread, and the dual mutant  has a modest defect, the acidic residues  appear to have additional functions. We have reported that mutagenesis of the Us9 dityrosine motif (PRV 440) also results in a complete loss of Us9 function Interestingly, the Us9 phospho-serines differ from adjacent negatively charged residues in the defect associated with their mutagenesis. Here, we report that loss of the adjacent four negatively charged residues in the acidic cluster ) results in a complete abrogation of anterograde spread in vivo, PRV expressing Us9 S51,53A does not spread to anterograde sites when compared to wild-type PRV Becker at equivalent early timepoints  were originally described as defective in vivo for anterograde spread compared to wild-type PRV at equivalent early time points in vivo, diserine mutant Us9 was found to undergo some anterograde spread, suggesting a kinetic defect in efficiency of transport. Our work with live cell imaging and chambered neuronal cultures has clarified this observation and supports this conclusion. The convergence of in vivo and in vitro experimentation therefore indicates a more subtle function for the Us9 diserine domain, such as the activation and/or stabilization of interactions with binding partners.Figure S1Detection of phosphorylated Us9 subspecies by the phospho-specific mouse monoclonal antibody clone 2D5E6. WB analysis of PK15 whole cell lysates 12 hours post-infection with PRV Becker, with or without CIP treatment, using rabbit polyclonal Us9 antibody and mouse monoclonal phospho-specific Us9 clone 2D5E6.(EPS)Click here for additional data file.Movie S1Live cell imaging of anterograde transport at 8 hours post-infection of dissociated SCG cultures.(MP4)Click here for additional data file.Movies S2Live cell imaging of anterograde transport at 8 hours post-infection of dissociated SCG cultures.(MP4)Click here for additional data file.Movie S3Live cell imaging of anterograde transport at 8 hours post-infection of dissociated SCG cultures. GFP, RFP, and Merged channels are shown in each movie, played back at 5 frames per second using 300 ms exposures in each channel. Anterograde directionality is to the right in each movie. (MP4)Click here for additional data file.


Brown adipocytes play an important role in regulating the balance of energy, and as such, there is a strong correlation between obesity and the amount of brown adipose tissue. Although the molecular mechanism underlying white adipocyte differentiation has been well characterized, brown adipocyte differentiation has not been studied extensively. Here, we investigate the potential role of dual-specificity phosphatase 10 (DUSP10) in brown adipocyte differentiation using primary brown preadipocytes.The expression of DUSP10 increased continuously after the brown adipocyte differentiation of mouse primary brown preadipocytes, whereas the phosphorylation of p38 was significantly upregulated at an early stage of differentiation followed by steep downregulation. The overexpression of DUSP10 induced a decrease in the level of p38 phosphorylation, resulting in lower lipid accumulation than that in cells overexpressing the inactive mutant DUSP10. The expression levels of several brown adipocyte markers such as PGC-1α, UCP1, and PRDM16 were also significantly reduced upon the ectopic expression of DUSP10. Furthermore, decreased mitochondrial DNA content was detected in cells expressing DUSP10. The results obtained upon treatment with the p38 inhibitor, SB203580, clearly indicated that the phosphorylation of p38 at an early stage is important in brown adipocyte differentiation. The effect of the p38 inhibitor was partially recovered by DUSP10 knockdown using RNAi.These results suggest that p38 phosphorylation is controlled by DUSP10 expression. Furthermore, p38 phosphorylation at an early stage is critical in brown adipocyte differentiation. Thus, the regulation of DUSP10 activity affects the efficiency of brown adipogenesis. Consequently, DUSP10 can be used as a novel target protein for the regulation of obesity. Adipose tissue is loose connective tissue that is primarily composed of adipocytes. It plays a critical role in energy homeostasis as both an energy reserve and an endocrine organ in animals et al. reported the positive role of p38 in adipogenesis in experiments using a p38 inhibitor et al. showed that bone morphogenic protein-2 (BMP-2) induced pluripotent mesenchymal cell lines C3H10T1/2 into the adipocyte lineage through p38 activation The p38 mitogen-activated protein kinases (MAPKs) are responsive to many stress stimuli such as cytokines, UV irradiation, and heat shock. They are involved in the processes of cell differentiation, apoptosis, and autophagy Protein phosphorylation is a common post-translational modification that regulates a wide range of the signaling pathways involved in differentiation, apoptosis, proliferation, gene regulation, and metabolism. Although phosphorylation at tyrosine occurs less frequently than phosphorylation at serine and threonine, tyrosine phosphorylation is very important because it occurs almost exclusively in multicellular eukaryotes All procedures used in animal experiments were performed according to a protocol approved by the Animal care and Use committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB).The pharmacological p38 inhibitor, SB203580, was acquired from Calbiochem. Antibodies for p38 (cat. no. 9212), phospho-p38  and DUSP10 (cat. no. 3483) were purchased from Cell Signaling. Anti-α-tubulin antibody was obtained from Sigma, and secondary antibodies were obtained from Abcam.2. Primary brown preadipocytes were induced to differentiate into mature brown adipocytes, as described previously 3] [all from Sigma]). They were then switched to a maintenance medium composed of DMEM, 10% FBS, 1 nM T3, and 20 nM insulin. The medium was replenished every 2 days.Brown preadipocytes were obtained from the interscapular BAT of mice  and isolated by collagenase dispersion as described previously Dusp10 gene was inserted into the multi-cloning site of the pRetroX-IRES-ZsGreen1 vector (Clontech). The catalytically inactive mutant of Dusp10, in which Cys-408 was replaced by Ser, was constructed by site-directed mutagenesis in plasmids To ensure that DUSP10 was stably expressed, a retrovirus-mediated infection system was used. The Dusp10 gene, as described by Sigma-Aldrich, and thus a sequence for inhibition of DUSP10 expression was obtained  was employed to knockdown DUSP10 in primary brown preadipocytes. Short-hairpin RNAs (shRNAs) were designed by selecting a target sequence specific to the mouse CTGCAATACCACG-3′). Following this, shRNA sequences were annealed and subcloned according to the manufacturer’s recommendations. The knockdown efficiency was approximately 50% in mouse primary brown preadipocytes. Non-targeting control shRNA  was obtained from Clontech.Total RNA was extracted from cultured cells with QIAzol lysis reagent , according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized using 2 µg of total RNA as the template, 500 ng of random primers, and cDNA synthesis kit components (Promega) in a total volume of 25 µL in accordance with the manufacturer’s recommendations Lipid droplets of differentiating or mature brown adipocytes were stained using the Oil red-O (Sigma) staining method, as described previously t-test. P values that exceeded 0.05 were regarded as statistically significant.Experimental differences were examined for statistical significance using Student’s 5′-GGATCCGAGCATCTTATCCA-3′, R: 5′-GGTGGTACTCCCGCTGTAAA-3′). The ND1 expression level was normalized to TBP (TATA box binding protein)  Mitochondrial DNA (mtDNA) in mature brown adipocytes was quantified using quantitative real-time PCR (qPCR) and Mito-Tracker fluorescence images. NADH dehydrogenase (ND1) primers were used to evaluate the mitochondrial DNA , and the mRNA levels were measured. As shown in DUSP10  inactivates its target kinases by dephosphorylating both the phosphoserine/threonine and phosphotyrosine residues. DUSP10 binds to and inactivates p38 and the N-terminal kinase, JNK, but not ERK Next, to test if the upregulation of DUSP10 was a phenomenon specific to brown adipogenesis, we conducted an expression analysis of DUSP10 during the white adipogenesis of 3T3-L1 preadipocytes. As shown in Dusp10 by using a retroviral system (Dusp10-IRES-GFP) to clarify the functional role of DUSP10 in brown adipogenesis. Dusp10-CS  and a control vector were used as negative controls. Infected cells were enriched using a FACS sorter. Most of the cells were found to be GFP positive upon examination by fluorescence microscopy, and the overexpression of wild-type and mutant DUSP10 was confirmed by a western blot analysis . MAPKs are key intracellular signaling pathways that play critical roles in many essential processes, including adipocyte differentiation. Thus, MKPs are subjected to tight regulation In this study, we performed a PTP profiling analysis during the brown adipogenesis of mouse primary brown preadipocytes. As expected, the results obtained from the PTP profiling analysis of brown adipocyte differentiation were significantly different from those for white adipocyte differentiation (data not shown). Among several PTPs identified as being differentially expressed during brown adipogenesis, we focused on the MAPK phosphatase (MKP) DUSP10. DUSP10  acts as a negative regulator of the stress-activated p38 and JNK MAPK kinases


We demonstrate that the present-day spatial distribution and orientation of the principal stresses within Larsen C ice shelf are akin to those within pre-collapse Larsen B. When Larsen B’s stabilizing frontal portion was lost in 1995, the unstable remaining shelf accelerated, crumbled and ultimately collapsed. We hypothesize that Larsen C ice shelf may suffer a similar fate if it were not stabilized by warm and mechanically soft marine ice, entrained within narrow suture zones.The collapses of the Larsen A and B ice shelves on the Antarctic Peninsula in 1995 and 2002 confirm the impact of southward-propagating climate warming in this region. Recent mass and dynamic changes of Larsen B’s southern neighbour Larsen C, the fourth largest ice shelf in Antarctica, may herald a similar instability. Here, using a validated ice-shelf model run in diagnostic mode, constrained by satellite and  Signs of instability in the Antarctic Larsen C ice shelf have raised concerns that it might soon collapse like its northern neighbour Larsen B. Kulessa et al. combine an ice-shelf model with satellite and geophysical data to show that despite dynamic similarities, Larsen C is presently stabilized by marine ice. The stability of an Antarctic ice shelf depends on the balance between the constructive stresses that ensure its integrity and the destructive stresses that compromise it. The spatial distribution and temporal evolution of the first and second principal stresses on Antarctic ice shelves depend on the geometry of the embayment in which the shelf is situated, the spatial and temporal attributes of the ice recharge it receives from feeding glaciers, environmental factors such as the evolution of atmospheric surface and oceanographic basal conditions2Fractures, such as horizontally extending rifts and vertically and horizontally extending surface and basal crevasses can be inherited from feeding glaciers. Fractures may also originate near the grounding line as the ice begins to float, or open up on the shelf in response to bending or shearing forces or thermal processes4We adopt our previous approachOur first stage of argument considers that both the first and the second principal stresses have highest absolute magnitudes near Larsen C’s grounding line and decrease non-uniformly towards the calving front . Stress-56Our second stage of argument considers that the distribution of stress-flow angles on Larsen B ice shelf in 1986 before its collapse -derived flow velocities of Larsen C ice shelf2021Our model sensitivity experiments with simulated softening of marine ice-bearing suture zones and major rifts on Larsen C ice shelf  reveal tSuture zones on Larsen C ice shelf tend to become thicker and narrower as they are advected downstream, owing to lateral compaction and the balance between continued accretion and basal melting2241328The presence of variably sized bodies of marine ice has already been inferred for several other Antarctic ice shelves, including the three largest, Filchner-Ronne18Our continuum-mechanical ice-shelf modelOur perturbation experiments implemented the most prominent marine ice-bearing suture zones and rifts on Larsen C ice shelf for the purpose of sensitivity testing . Adoptin−1 based on common-midpoint surveys conducted in both field seasons4The GPR data presented in Our seismic reflection data were acquired in walk-away mode in 2008–09 ref.  using a B.K. and A.J.L. respectively led NERC projects NE/E012914/1 and NE/I016678/1 that made this work possible; and D.J. was the post-doctoral scientist for both. E.C.K. and P.R.S. were co-investigators on NE/E012914/1. D.J. implemented and conducted the ice-shelf model runs presented here. Building on E.C.K.’s expertise, B.K. and A.J.L. collected the field geophysical data used as model constraints; and B.K., E.C.K. and D.J. processed them. A.J.L. and D.J. acquired and processed the satellite data used as model constraints. P.R.S. contributed on the mechanical properties of and fracture processes operating on ice shelves.How to cite this article: Kulessa, B. et al. Marine ice regulates the future stability of a large Antarctic ice shelf. Nat. Commun. 5:3707 doi: 10.1038/ncomms4707 (2014).Supplementary Figures 1-3


Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect. Autophagy is a cellular process responsible for the degradation of cytoplasmic components via the lysosomal pathway During the formation of the autophagosome cytosolic microtubule-associated protein 1 light chain 3-I (LC3I) is cleaved and conjugated with phosphatidylethanolamine (PE), leading to formation of LC3II, an autophagic vacuole-associated form Streptomyces griseus strains, specifically inhibits vacuolar H+ ATPase (V-ATPase), which plays a role in the acidification of the lysosome leading to the digestive function of the lysosome Autophagy is a complicated process that consists of autophagosome biogenesis, maturation, and fusion with lysosomes. A number of diverse signaling complexes have effects on the respective molecular steps in the regulation of the autophagic process N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) on the arrest of the autophagic process through the inhibition of lysosomal fusion in normal rat liver Ac2F cells. MHY1485 was synthesized based on its morpholino triazine structure that is known to bind mTOR  with reference to the respective residual solvent or deuteriated peaks . Coupling constants are reported in hertz. The abbreviations used are as follows: s (singlet), d (doublet), m (multiplet). The electron-impact ionized mass spectra (EIMS) were obtained at an electron beam energy of 75 eV. All the reactions described below were performed under nitrogen or argon atmosphere and monitored by thin-layer chromatography (TLC). TLC was performed on Merck precoated 60 F254 plates. Column chromatography was performed using silica gel 60 . All anhydrous solvents were distilled over CaH2 or Na/benzophenone prior to use.All reagents were purchased from Aldrich and were used without any further purification. Melting points are uncorrected. Nuclear Magnetic Resonance (NMR) data were recorded on a Varian Unity INOVA 400 spectrometer and Varian Unity AS 500 spectrometer, using CDCl1H and 13C NMR experiments.To a stirred solution of cyanuric chloride  in acetone (30 mL) and crushed ice, a mixture of triethylamine  and morpholine  was added at −10°C. After the addition, the reaction mixture was stirred at room temperature for 1 h and diluted with 50 mL of water. The white solid generated was filtered and washed with water and acetone. The white solid was dried under reduced pressure . The final product precipitated from methanol washing needed no further purification as its purity was ascertained by TLC using a multiple developments method and 1H NMR  δ 3.74–3.67 ; 13C NMR  δ 169.8 , 164.7 (C6), 66.9 (4×OCH2), 44.1 (4×NCH2); IR (KBr): 1504, 1355, 1122, 806 cm−1; Mass (EIMS), 285 (M)+.Melting point (m.p): 172.6–173.9°C; A suspension of 4,4′-dimorpholine , 4-nitroaniline , and potassium carbonate  in N,N-dimethylformamide (2 mL) was refluxed for 5 h. The precipitates generated were filtered on a Buchner funnel and washed methanol and water. After being dried under reduced pressure, the title product  was obtained as a pale yellow solid.1H NMR  δ 8.20 , 7.70 , 7.02 , 3.81–3.79 , 3.76–3.74 ; 13C NMR  δ 165.2 , 164.6 (C2), 147.8 (C1′), 141.1 (C4′), 125.5 , 119.1 , 66.6 (4×OCH2), 44.1 (4×NCH2); IR (KBr): 3368, 1535, 1502, 1338, 1111, 851 cm−1.m.p: 265.0–266.2°C; l-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum . Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2/95% air. Under starvation conditions, cells were treated with Hank’s Balanced Salt Solution (HBSS) . Our compound was treated under the same conditions as the growth medium to exclude the effect of serum reduction to induce autophagy. For assessment of autophagic flux, the lysosomotropic agents bafilomycin and chloroquine were applied 1 h before the cell harvest at concentrations of 10 nM and 100 µM, respectively Rat hepatocytes, Ac2F cells were obtained from ATCC . The cells were grown in Dulbecco’s modified eagle medium  containing 2 mM Cells were washed with cold PBS and harvested. Cell lysates were prepared using RIPA buffer containing 50 mM Tris-HCl (pH), 150 mM NaCl, 1% NP-40, 1 mM DTT, 0.1 mM NaF, 1 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Protein concentration was determined by the bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as a standard. Equal amounts of protein were separated on 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The gels were subsequently transferred onto a polyvinylidene difluoride membrane  by electroblotting for 2 h at 60–75 V. The membranes were blocked in a 5% nonfat milk solution in Tris-buffered saline (TBS) with 0.5% Tween-20, and incubated with primary antibodies as indicated. Pre-stained protein markers were used for molecular-weight determination.?5 cells were seeded in coverglass-bottom-dish, incubated overnight, and then transfected with the adenovirus encoding green fluorescent protein-microtubule-associated protein 1 light chain 3  at a concentration of 1,000 virus particles/cell in DMEM. After incubation for 24 h, cells were treated with compounds or starved. For visualization of lysosomes, cells were incubated with LysoTracker® at a concentration of 60 nM for 1 h. Confocal images were obtained with FV10i FLUOVIEW Confocal Microscope .Approximately 1×10http://swissmodel.expasy.org/qmean). In order to prepare for docking simulation, we performed deleting solvent, adding hydrogen, adding charge, and replacing incomplete side chain of the structure model using the Chimera program (http://www.cgl.ucsf.edu/chimera/). To add charge, we used the charge data of AMBER ff99SB for standard residues.A three-dimensional model of mTOR (NCBI GI number : 4826730) comprising 2,549 amino acids was built using SWISS-MODEL program in silico protein-ligand docking, AutoDock4.2 is the most commonly used because of its automated docking capability http://www.cambridgesoft.com).For docking simulation, we used the AutoDock 4.2 program according to the software’s manual. Among the many tools available for P values of <0.05 were considered statistically significant.ANOVA was used to analyze differences among all groups. Differences in the means of individual groups were assessed by Fisher’s protected least significant difference post hoc test. First, we evaluated the LC3II/LC3I ratio in normal rat liver Ac2F cells treated with MHY1485 with different doses and times. MHY1485 markedly increased the LC3II/LC3I ratio dose-dependently and time-dependently . As showTo confirm that the increased LC3II/LC3I ratio was an outcome of the accumulation of autophagosomes and not elevated autophagic activity, we measured autophagic flux with the lysosome inhibitors bafilomycin A1 and chloroquine Next, to confirm the inhibitory effect of MHY1485 on autophagy, we evaluated the autophagic flux and the colocalization of LC3-positive vacuoles and lysosomes using confocal microscopy in cells stimulated by starvation, which is a powerful circumstance leading to autophagy. Active autophagic processes occurred in cells with starvation, as demonstrated by increased accumulation of LC3II protein after treatment of bafilomycin A1 or chloroquine. However, the extent of accumulated LC3II protein was less after treatment of bafilomycin A1 or chloroquine in the MHY1485 treated starved cells . These dmTOR, a major regulator of autophagy, plays a role in the inhibition of autophagy through the phosphorylation of unc-51-like kinase 1 (ULK1), a component involved in the initiation of autophagic process Autophagy is a self-degradation process via the lysosomal pathway that is required for the normal turnover of cellular components and the starvation response It has been reported that autophagic markers such as LC3 and beclin-1 increase when the fusion between autophagosomes and lysosmes is blocked Many small molecules that induce the autophagic process including rapamycin, SAHA, temozolomide and curcumin have been explored One interesting finding is the activation of mTOR by MHY1485. mTOR is a major regulator of initiation of autophagy In conclusion, we found a novel mechanism for inhibition of autophagy by MHY1485. The effects of the synthetic compound MHY1485 included the inhibition of autolysosome formation, leading to the arrest of autophagy and the induction of mTOR activity.


The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.558A and pcDNA3/HA-moesinT558D were applied in endothelial cells.Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinTThe results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction. Advanced glycation end products (AGEs) are a heterogeneous group of complex compounds that are formed irreversibly in serum and tissues via a chain of non-enzymatic chemical reactions . The rolSince ROCK does not directly act on F-actin and other cytoskeletal molecules, there is a missing link between the activation of ROCK and the cytoskeleton reorganization. ERM (ezrin/radixin/moesin) proteins are emerging as the potential candidates that likely mediate this process. Serving as cross-linkers between actin filaments and plasma membrane, ERM molecules are engaged in cell adhesion, microvilli formation, cell motility, etc -23. Moes558A and pcDNA3/HA-moesinT558D were applied in endothelial cells and the AGE-stimulated responses were compared.One of the purposes of this study was to further investigate the specific effect of RhoA/ROCK pathway on endothelial responses to AGEs. By transfection of recombinant adenoviruses targeting RhoA activity and usage of a more specific ROCK inhibitor, we explored the contribution of this signaling pathway to the AGE-induced increase of endothelial monolayer permeability and alterations of the F-actin cytoskeleton by clarifying the phosphorylation state of RhoA and ROCK after exposure to AGEs. This present study is also aimed to further confirm the interaction of ROCK and its downstream target moesin by using Co-immunoprecipitation (Co-IP). To identify the AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinTA G-LISA™ RhoA activation assay kit was purchased from Cytoskeleton . Antibody recognizing total ROCK, ROCK inhibitor H-1152, and protein G plus/protein A agarose suspension were obtained from Calbiochem (USA), while antibody recognizing p-ROCK came from Upstate . RhoA N19 recombinant adenovirus (dominant negative) and RhoA L63 recombinant adenovirus (constitutively active) were purchased from Cell Biolabs . Antibody recognizing moesin was purchased from Abcam  while anti-phosphorylated (p-)moesin (Thr558) antibody was purchased from Santa Cruz Biotechnology . Antibody recognizing HA was purchased from Cell Signaling Technology . Lipofectamine™ LTX and PLUS™ reagents were obtained from Invitrogen Technology. Primers were designed using Primer Premier 5 software and synthesized by Invitrogen (USA). Restriction enzymes EcoR I and Xho I were from Takara (Japan). High Fidelity DNA polymerase KOD-Plus, KOD-Plus-Mutagenesis kit and Thunderbird SYBR qPCR Mix were obtained from Toyobo (Japan). Rhodamine-phalloidin was obtained from Molecular Probe . MCDB 191 medium, DMEM medium, fetal bovine serum (FBS), trypsin, glutamine, penicillin, and streptomycin were all from Gibco BRL . Chemicals were purchased from Sigma  unless otherwise indicated.Advanced glycation end product-modified human serum albumin (AGE-HSA) was prepared as previously reported , essenti2. Primary human umbilical endothelial cells (HUVECs) (from Sciencell) were maintained in DMEM containing 10% FBS at 37°C in a humidified atmosphere with 5% CO2.A human dermal microvascular endothelial cell (HMVEC) line was purchased from Cell Applications  . Cells wIn all experiments, HMVECs were grown to 90% confluence and starved of serum for 2 hours before being stimulated with AGE-HSA at the indicated doses and for the indicated times. The AGE-HSA concentrations used in this experiment were based on data from our previous studies of HMVECs or freshly cultured primary HUVECs ,20. To ccell lysis buffer with protease inhibitors, and centrifugating at 14,000 rpm at 4°C for 2 min to remove cell debris. The protein concentration was determined according to the manufacturer's protocol, and cell extracts were equalized to a protein concentration of 1.5 mg/ml for assay. Luminescence was detected according to the manufacturer's recommendations with minor modifications after incubation overnight at 4°C with the primary anti-RhoA antibody.RhoA activity was determined by using a luminescence-based G-LISA™ RhoA activation assay kit  according to the manufacturer's instructions. This assay employs a Rho-GTP-binding protein coating the wells of a 96-well plate. Active, GTP-bound Rho in cell lysates binds to the wells, while inactive GDP-bound Rho is removed through the washing steps. Then the bound active RhoA is detected by incubation with a specific RhoA antibody followed by an HRP-conjugated secondary antibody and a detection reagent, after which the luminescence is read on a microplate luminescence reader . HMVECs were cultured in 6-well plates and treated as indicated above. Proteins were harvested by incubating the provided 3VO4) with protease and phosphatase inhibitors. Samples were subjected to SDS-PAGE, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Blots were blocked with 5% bovine serum albumin in TBS containing 0.5% Tween 20 (TBS-T) for 1 h and then incubated with an 1:1000 dilution of primary antibody for p-ROCK  or p-moesin  overnight at 4°C on a rocker. After three washes for 5 min each with TBS-T, the blots were incubated with an 1:1000 dilution of HRP-conjugated species-specific respective secondary antibody for 1 h at room temperature. After washing three times for 5 min each with TBS-T, protein bands were visualized by chemiluminescence and then densitometric analysis was done by using Kodak IS2000R Imaging Station.Total cellular extracts were prepared by lysis and sonication of the cells in lysis buffer  ,30. The The transfection of plasmids was carried out by endofree plasmid midiprep kit. HUVECs of about 90%~95% confluent in 500 μl Opti-MEM medium without antibiotics were transfected with plasmids encoding HA-tagged two mutant forms of moesin (T558A and T558D) using Lipofectamine™ LTX and PLUS™ reagents (Invitrogen) according to the manufacturer's instructions. Briefly, for a 6-well format, 1 μg DNA was incubated in 500 μl Opti-MEM (antibiotics-free) while 8 μl lipofectamine LTX and 2 μl plus reagent were added and left at room temperature for 30 min. The cultured cells were washed once with Opti-MEM. The DNA-lipid complexes were added to the plates and incubated for 24 h, followed by stimulation with AGE-HSA . The cells were then used for Realtime PCR, immunoblotting, endothelial monolayer permeability assay, or immunofluorescence, respectively.Total RNA was isolated from HUVECs by total RNA extraction kit (Biomegia USA) according to the manufacturer's protocol. Realtime PCR amplification was performed with moesin-specific primers. Total RNA of 1 μg was used as templates for cDNA synthesis in the reverse transcriptase reaction. After an initial 60 s predenature and 15 s denaturation at 95°C, cDNA was amplified for 40 cycles and then annealed at 60°C for 60 s for moesin and GAPDH. Under optimized conditions there was a single melting curve and no primer-dimer formation. The copy number for each mRNA was determined using a standard curve generated with external standards of known copy number. All the primers were designed using Primer Premier 5 software  after stimulated with AGE-HSA (50 mg/L for 1 h). Cells were lysed in the lysis buffer and the lysates were centrifuged at 10,000 g for 10 min at 4°C. Supernatants were incubated overnight at 4°C with ROCK antibody, moesin antibody or p-ROCK antibody, respectively, and then incubated with Protein A/G PLUS-Agarose  for 3 h at 42, V is the volume of the abluminal chamber, and [L] is the luminal albumin concentration.Endothelial monolayer permeability was measured as described by Tinsley . ECs werECs were plated on gelatin-coated glass-bottomed microwell plates  and cultured until confluence. After appropriate treatment, the cells were fixed and permeabilized for 15 min at room temperature in PBS with 3.7% formaldehyde and 0.5% Triton X-100. After a thorough wash with PBS, cells were incubated with rhodamine-phalloidin  for 40 min at room temperature. Then the cells were washed three times with PBS, and the plate was mounted for observation and imaging with a Leica TCS SP2 laser confocal scanning microscope .Data were normalized to control values and are reported as a percentage of the baseline values (mean ± SD) for at least three independent experiments. Results were analyzed by one-way ANOVA followed by post hoc comparison. The level of significance was set at P < 0.05.P < 0.01 compared with the control)  Figure . AGE-HSA) Figure . In contP < 0.05 vs. control)  Figure  and 4B. ) Figure  and 4D. P < 0.05)   Figure . Transfe) Figure , along w) Figure .It was indicated that down-regulation of RhoA activity by transfection of HMVECs with RhoA N19 abolished the increase of HMVEC monolayer permeability induced by AGE-HSA treatment Figure . FormatiOur previous study showed that inhibition of ROCK activation by Y-27632 could suppress the phosphorylation of moesin induced by AGE-HSA and block the AGE-evoked increase of HMVEC monolayer permeability . H-1152,P < 0.05)  Figure . The up-To further verify the relationship of ROCK and moesin, the direct interaction of endogenous ROCK and moesin after AGE-HSA stimulation was assessed using Immunoprecipitation (IP) - Immunoblotting (IB) analysis. HUVEC extract was obtained after stimulation with 50 mg/L AGE-HSA for 1 h. Physical interaction between ROCK and moesin was confirmed in HUVECs with or without AGE-HSA application Figure . The binT558A) as inhibited mutant  as activated mutant  from 158.34 ± 4.17% to 116.67 ± 3.21%. It also preserved the distribution of F-actin in endothelial cortex area after AGE-HSA stimulation. The transfection of plasmid pcDNA3/HA-moesinT558D itself increased endothelial monolayer permeability to 152.62 ± 7.14%, also triggered the formation of F-actin stress fiber H and reactive oxygen species, p38 MAPK, phospholipase C, and calmodulin-dependent protein kinase ,20,32,38It is necessary to extend the downstream target of Rho/ROCK since ROCK did not directly interacts with F-actin. It has been well documented that activation of RhoA/ROCK can phosphorylate and inactivate myosin light chain phosphatase (MLCP), hence promoting MLC phosphorylation and acto-myosin contraction in endothelial cells -47. AnotPreviously, we have demonstrated that the inhibition of ROCK with Y-27632 attenuated the AGE-induced phosphorylation of moesin in HMVECs . The datT558A was introduced to HUVECs, the AGE-induced phosphorylation of moesin was attenuated (Figure T558D mimicked the AGE-evoked endothelial response with formation of F-actin stress fiber and increase of endothelial monolayer permeability. These data are consistent with the results from Zhou et al, indicating that exogenously expressed meosin bears biochemical characteristics similar to endogenous protein [It has been revealed that tyrosine and threonine residues are major phosphorylation sites in ERM protein ,50,51. Bd Figure , as welld Figure . The act protein . AlthougTaking together, this study provides direct evidences to show that the up-regulation of RhoA activity is involved in AGE-induced endothelial dysfunction. ROCK physically interacts with moesin and the RhoA-triggered phosphorylation of ROCK results in phosphorylation of threonine at 558 residue in moesin. This RhoA/ROCK -dependent moesin phosphorylation regulates AGE-induced endothelial dysfunction.AGE: Advanced glycation end products; AGE-HSA: AGE-modified human serum albumin; DMSO: Dimethylsulfoxide; EC: Endothelial cell; ERM: Ezrin/radixin/moesin; FBS: Fetal bovine serum; HMVEC: Human dermal microvascular endothelial cell; HSA: Human serum albumin; HUVECs: Human umbilical vein endothelial cells; MAPK: Mitogen activated protein kinase; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; QPCR: Real-time quantitative PCR; RAGE: Receptor of AGE; Rho: Ras homologue oncogene; RNA: ribonucleic acid; ROCK: Rho kinase; RT-PCR: reverse transcription polymerase chain reaction.The authors declare that they have no competing interests.QH conceived the study, arranged the collaboration, initiated the manuscript, edited and compiled the final version for submission. JW and HL participated in its design and coordination. JW, HL, BCh, QL, XH, LW, XG performed Laboratory analyses and study design. All authors read and approved the final manuscript.


This pathway is likely to be central to EGFR-mediated trophoblast survival. Furthermore, EGF treatment induces proliferation and inhibits apoptosis, while Iressa induces apoptosis.We identified a major peptide signaling target of EGF/EGFR pathway and explored the consequences of blocking or activating this pathway in the first trimester extravillous trophoblast cells, HTR-8/SVneo. A global analysis of protein phosphorylation was undertaken using novel technology (Kinexus Kinetworks) that utilizes SDS-polyacrylamide minigel electrophoresis and multi-lane immunoblotting to permit specific and semiquantitative detection of multiple phosphoproteins. Forty-seven protein phosphorylation sites were queried, and the results reported based on relative phosphorylation at each site. EGF- and Iressa- treated HTR-8/SVneo cells were subjected to immunoblotting and flow cytometry to confirm the phosphoprotein screen and to assess the effects of EGF versus Iressa on cell cycle and apoptosis. EGFR mediates the phosphorylation of important signaling proteins, including PKB Epidermal growth factor (EGF) and its receptor (EGFR) are known to play critical roles in cell growth, differentiation, angiogenesis, and inhibition of apoptosis. EGF is a multifunctional 53 amino-acid peptide known for its ability to stimulate proliferation and differentiation in the placenta as well as many other cells and tissues . EGF is EGFR signals through a series of protein kinases. These are important transducers of information in human cells with the ability to regulate cellular functions by catalyzing the direct phosphorylation of target proteins . ProteinμM, Astra-Zeneca Pharmaceuticals, London, England) for 20 h, and Iressa plus EGF. Iressa was dissolved in DMSO and EGF was dissolved in distilled water according to the manufacturer's protocol. Controls were performed to ensure the absence of effects with the respective vehicles. Transformed human trophoblast-8 (HTR-8/SVneo) cells, originally obtained from Professor Charles Graham , were grown to approximately 80% confluence in minimum essential medium  with 50 mL of fetal bovine serum  . The celμg of protein was transferred to a fresh 1.5 mL Eppendorf screw cap vial and mixed with 4x sample buffer  in a ratio of 1 : 4 sample to buffer. Samples were then boiled for 4 min, placed in tightly locked tubes and shipped to Kinexus, Inc. for quantification. Cells were harvested and washed twice with PBS. Protein lysate was obtained according to Kinetworks' guidelines . Briefly, protein was extracted using 0.5% Triton X-100 lysis buffer with sonication for 10 seconds on ice to rupture the cells. Cell extracts were then ultra-centrifuged for 30 min at 100,000 ×g. The supernatant was transferred to fresh tubes, and protein concentrations were assayed promptly using Bradford dye and a microplate reader according to the manufacturer's protocol . For each sample, 400 Multipeptide immunoblotting was performed according to the manufacturer's instructions using Kinetworks Phospho-Site Screen 1.3 , which tracks the abundance of the following phosphoprotein targets: Adducin A (S724), Adducin G (S662), CDK1 (Y15), CREB (S133), ERK1 (T202/Y204), ERK2 (T185/Y187), GSK3-alpha , GSK3-beta , JUN(S73), JNK/SAPK (T183/Y185), MEK1/2 (S217/S221), MEK3/6 (S189/S207), MSK1 (S376), NR1 (S896), p38-alpha MAP kinase (T180/Y182), S6K p70 (T389), PKB-alpha/AKT (S473/T308), PKC-alpha (S657), PKC-alpha/beta (T638/641), PKC-delta (T507), PKC-epsilon (S729), PKR (T451), RAF1 (S259), RB1 , RSK1 (T359/S365), JNK/SAPK (T183/Y185), SMAD1 (S463/465), SRC (Y418/Y529), STAT1 (Y701), STAT3 (S727), and STAT5 (Y694). By this method, 47 phosphosites on 31 proteins are identified. μg of total protein extract resolved on a 13% single-lane SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Using a 20-lane multiblotter from BioRad , the membrane was incubated with different mixtures of up to three antibodies per lane that react with a distinct subset of phosphorylated cell signaling proteins of known molecular masses, as listed above. After further incubation with a mixture of relevant HRP-conjugated secondary antibodies , the blots were developed using ECL Plus reagent , and the signals were quantified using Quantity One software (BioRad)  as previously described by Lange, et al. , whereas five target phosphoproteins  were downregulated. For cells treated with Iressa alone, six phosphoproteins  were upregulated and 11 phosphoproteins  were downregulated. Representative blots of cells grown under control conditions, compared to cells treated with EGF, Iressa, and EGF + Iressa are shown in Figures Using Kinetworks Phospho-Site Screen 1.3, tracking the 47 phosphorylation targets specified in the methods, multiple differences were noted in the phosphoprotein profile of each treatment group using the cutoff of at least 25% difference between treatments. Compared to controls, EGF treated cells demonstrated the upregulation of 12 phosphoproteins [Adducinα/AKT consistently met these criteria, both at S473 and T308. Phosphorylation at S473 is shown in bar graph format in α/AKT S473 with EGF treatment but downregulation with Iressa alone or Iressa + EGF. We reasoned that the principal EGFR phosphorylation target(s) would be both robustly induced by EGF and strongly downregulated by Iressa. Protein Kinase B-α/AKT), and some were not. Other sites were phosphorylated but were not influenced by EGFR activation or inhibition. Proteins that were phosphorylated in HTR-8/SVneo cells but not significantly affected by EGFR activation or blockade, include the following: Adducin a, Adducin g, CDK1, CREB, ERK1, ERK2, GSK3a, GSK3b, JUN, MEK1/2, MEK3/6, MSK1/2, NR1, PKCa, PKCa/b, PKCe, RAF1, SAPK, SRC, and STAT1 . These sFlow cytometry was employed to assess the effects of EGF +/− Iressa and Iressa alone on the cell cycle Figures . CompareμM Iressa enhanced cellular apoptosis to 16.5% . Addition of Iressa to serum-depleted media resulted in a further significant increase in the percent of apoptotic cells (P = 0.038). EGF was able to partially inhibit apoptosis compared to serum depletion although this effect did not reach statistical significance in this assay and is thus not shown. The effect of Iressa on cell viability confirmed the apoptotic assays, where treatment resulted in a 31% decrease in HTR-8 cell viability compared to the serum-starved condition (data not shown). The effects of Iressa on apoptosis were studied further using fluorescence microscopy in the presence of Hoechst dye, as shown in to 16.5% . Using tThe literature strongly supports a primary role for EGFR signaling in the development and the function of the trophoblast . The EGFThis study utilized a novel technology to analyze protein expression and phosphorylation changes in 31 proteins. The pathways queried are illustrated in α/AKT. Two sites on this protein, S473 and T308, were controlled by EGFR signaling. PKBα/AKT phosphorylation occurs through phosphatidylinositol 3 kinases. Full activation of PKBα/AKT requires phosphorylation at both S473 and T308. These sites are independently phosphorylated by two intermediates, PIP3 and PIP2, respectively [α/AKT modulates the phosphorylation of over 40 different downstream peptides. PKBα/AKT is involved in the regulation of many biological processes, including cell proliferation, growth, apoptosis, and tumorigenesis, as shown in α/AKT is particularly well known as a negative regulator of apoptosis [α/AKT inhibits JNK activation, leading to the inability to activate the cellular signaling scaffold required to induce apoptosis (17). PKBα/AKT has also been implicated in nitric oxide regulation, glucose homeostasis, and insulin signaling [α/AKT and the consequences on downstream signaling molecules may have detrimental effects on newborn pregnant mice. Gene disruption of PKBα/AKT results in placental insufficiency, fetal growth impairment, and death in the newborn [The principal EGFR target reproducibly induced by EGF and inhibited by Iressa was PKBectively . Interespoptosis . Phosphoignaling –18. Stud newborn .α/AKT as a principal target, and blocking this pathway with the EGFR-specific tyrosine kinase inhibitor, Iressa, results in programmed cell death. We hypothesize that blocking EGFR signaling may result in apoptosis via inhibition of PKBα/AKT. Loss or deactivation of PKBα/AKT, as suggested by gene disruption models, may be linked to the pathogenesis of IUGR. Further studies are planned to determine whether IUGR in human pregnancies is linked to enhanced trophoblast apoptosis in response to the dephosphorylation of PKBα/AKT downstream of EGFR. In summary, these studies indicate that EGF signaling through EGFR activates PKB


The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely. The polypeptide chain initiation factor eIF4E plays an important role in regulating the translation of capped mRNAs in eukaryotic cells and it is widely accepted that the availability of eIF4E to form the eIF4F initiation complex  can be a rate-limiting step in the initiation of protein synthesis bone fide oncogene product Initiation factor eIF4E is now considered to be a The ability of the 4E-BPs to bind eIF4E is regulated by their state of phosphorylation, which in turn is controlled by the protein kinase mammalian target of rapamycin (mTOR). The latter exists in two complexes, mTORC1 and mTORC2, and is important in the control of a wide variety of pathways in health and disease 7GTP-Sepharose beads were from GE Healthcare. [35S]methionine was supplied by MP Biomedicals . Antibodies against total 4E-BP1 and phosphorylated 4E-BP1 (Ser64) were from Santa Cruz Biotechnology Inc.  and Cell Signalling Technology  respectively. Antibodies against total Akt and phosphorylated Akt (Ser473) and against total p70S6K and phosphorylated p70S6K (Thr389 and Thr421/Ser424) were from Cell Signalling Technology. The antibody against GAPDH was from Merck Millipore . Anti-eIF4GI was produced in-house. Horseradish peroxidise-linked secondary antibodies were from Cell Signalling Technology. PVDF membrane and rainbow markers were supplied by GE Healthcare.Tissue culture materials were from Gibco Life Technologies Ltd.  and GE Healthcare . The mTOR inhibitors Ku-0063794, rapamycin, PP242 and PI-103 were from Tocris Bioscience (Bristol U.K.). mMouse embryonic fibroblasts (MEFs) with a double knockout of the 4E-BP1 and 4E-BP2 genes 2 in Dulbecco’s modified Eagle medium supplemented with penicillin (50 units/ml), streptomycin (50 units/ml) and 10% foetal bovine serum. Cultures were split every 3–5 days and all experiments were performed on cells that were in exponential growth. To induce hypertonic stress the cells were incubated for 1 h in the above medium containing additional NaCl (0.1 M except where otherwise indicated). For experiments involving serum deprivation the cells were washed with phosphate buffered saline (PBS) and then incubated in the above medium with or without serum for 24 h. The cells were then incubated without or with rapamycin (100 nM), Ku-0063794 (1 µM), PP242 (5 µM) or PI-103 (5 µM) for 1 h.The cells were maintained in monolayer cultures at 37°C in humidified air with 5% CO35S] methionine (1–2 µCi/ml for 1 h) into trichloroacetic acid-insoluble material as described previously Following the treatments described above overall protein synthesis in intact cells was measured by the incorporation of [Cells were washed in PBS and subjected to lysis as previously described 7GTP-Sepharose beads as described Initiation factor eIF4E and its associated proteins were isolated from cell extracts  by affinity chromatography on mThe data from the protein synthesis determinations are shown as the means ± S.E.M. Independent experiments were performed at least three times and typical examples are presented. Unpaired t tests  were used to determine statistical significance and p values of <0.05 are considered to demonstrate significant differences.It is well established that the stress imposed on cells by mildly hypertonic conditions results in a marked inhibition of protein synthesis. However the consequences of inhibition of mTOR activity under such conditions have not previously been examined. Accordingly we investigated the effect of the mTOR inhibitor Ku-0063794 on translation in murine embryonic fibroblasts in the presence of increasing salt concentrations. 35S]methionine incorporation, are summarized in To determine the requirement for the 4E-BPs for the effects of Ku-0063794 on protein synthesis under normal and hypertonic conditions, similar experiments were performed on 4E-BP1/2 double knockout (DKO) cells. The latter cells are genetically disrupted for both 4E-BP1 and 4E-BP2 and express neither of these proteins Similar experiments were carried out using MEFs with a serine to alanine mutation at position 51 of the α subunit of initiation factor eIF2 (S51A cells). These cells are unable to undergo phosphorylation at this site in response to a variety of physiological stresses, rendering them deficient in the regulation of polypeptide chain initiation 35S]methionine incorporation at all under normal conditions but reduced protein synthesis by 29% in 4E-BP wild-type cells under hypertonic conditions   . As notep<0.002) . SimilarAnother compound that inhibits not only mTORC1/2 but also PI3-kinase is the pyridofuropyrimidine PI-103 64 phosphorylation site. This is consistent with evidence that mTOR remains active under hypertonic conditions and indeed may be activated by osmotic stress Since 4E-BP1, when in a hypophosphorylated state, regulates cap-dependent translation by competing with eIF4G for binding to eIF4E, it was important to determine whether hypertonic conditions influence the state of phosphorylation of this protein. 64, and was dephosphorylated in the presence of Ku-0063794, it may correspond to 4E-BP3. There was no 4E-BP signal under any conditions when eIF4E and its associated proteins in DKO cells were analysed by immunoblotting  of 4E-BP3 is not sufficient to compensate for the lack of 4E-BP1 and 4E-BP2 in sensitizing cells to mTOR inhibition. In addition, our data show that the decreased phosphorylations of p70S6K and Akt that occur when mTORC1/2 activity is inhibited are not sufficient to affect overall protein synthesis, at least in the short term. Thus these proteins (as well as other mTOR substrates in vivo due to lack of oxygen and nutrient supplies and malignant cells can evolve strategies to overcome such adverse conditions. In view of the importance of the 4E-BPs for the control of proliferation of untransformed cells, as well as the well known role of dysregulation of the eIF4E/4E-BP system in cancer progression Recent reports using cell lines in which the levels of 4E-BP1 and 4E-BP2 have been experimentally manipulated show that these proteins play important roles in the regulation of cell proliferation


Saccharomyces cerevisiae (yeast) strain National Collection of Type Culture (NCTC) 1071606 as the test organism. From the standard curve the yeast inhibitory potencies of the shampoos in nystatin international unit equivalents were determined from the respective inhibition zones of the test samples of the shampoos. Under test conditions four shampoo samples showed remarkable fungal inhibitory potencies of 10227, 10731, 12396 and 18211 nystatin international unit equivalents/ml while two shampoo samples had extremely feeble inhibitory potencies 4.07 and 4.37 nystatin international unit equivalents/ml although the latter two products claimed antifungal activity. The potency determination method could be applied to any antidandruff shampoo with any one or a combination of active ingredients.A convenient standard microbiological potency determination test for the antidandruff shampoos was developed by adopting the pharmacopoeial microbiological assay procedure of the drug nystatin. A standard curve was drawn consisting of the inhibition zone diameters vs. logarithm of nystatin concentrations in international units using the fungus  Malassezia also known as Pityrosporum, which is a tiny yeastShampoos may be classified into three distinct categories. Plain detergent shampoos that are more like soaps in their properties and uses, the cosmetic shampoos with conditioners, dyes or other cosmetic ingredients used for cosmetic purposes and the medicated shampoos, which are formulated to contain any drug substance as in the case of antidandruff shampoos. Each such shampoo category must be dealt accordingly for regulatory purposes. Medicated antidandruff shampoos containing typical drugs such as climbazole1, A2 and A334Saccharomyces cerevisiae as the test organism. It was thought that this procedure could be adopted to screen the shampoos claiming antidandruff activity since a fungus is also responsible for infections associated with dandruff. The proposed microbiological assay method is further justified as both nystatin and the antifungal shampoos are aimed at treating superficial fungal infectionsThe antifungal drug nystatin consists of a mixture of biologically active substances consisting of tetraene molecules ASix brands of shampoos claiming antidandruff activity were procured from the market. Three of them contained zinc pyrithioneSaccharomyces cerevisiae strain NCTC 1071606 was used as the inoculating agent in the Medium F as per the pharmacopoeia. The inoculum was adjusted to contain 10Nystatin standard serial dilutions containing 200, 100, 50 and 25 international units per milliliter were prepared in pH 6 buffer containing 5% dimethylformamide as per the British Pharmacopoeia. The inhibition zones for standard nystatin solutions were determined by incubating the inoculated petri dishes containing 100 μl nystatin standard dilutions per well at 30° for 18 h. Centre well of each Petri dish was filled with 100 μl of the blank buffer solution as the control. For each dilution inhibition zones were determined in triplicate and three diameters for each inhibition zone were recorded. Prior to the main analysis serial dilutions of the shampoo samples were prepared aseptically with the pH 6 buffer containing 5% dimethylformamide and the dilutions corresponding to inhibition zones falling within the range of the standard curve were determined. Four shampoo samples had to be diluted 250 times and two were tested without diluting. Inhibition zones for each shampoo sample were also determined in triplicate using 100 μl per well and three diameters for each inhibition zone were recorded. The centre well was filled with 100 μl of the blank buffer solution as the control. The main potency determination assay was carried out simultaneously for the nystatin standard curve and the appropriately diluted shampoo samples using the same culture media lot under the same conditions.Since the antidandruff shampoos have diverse active ingredients it is difficult to test each one of them requiring different analytical methods. The study was designed to evaluate any of the shampoos with respect to an equivalent in terms of a reference material. Nystatin was selected as the reference antifungal agent since a microbiological antifungal assay for this substance is prescribed in the British Pharmacopoeia. For a given shampoo with any one of the antifungal agents the fungal inhibitory potency will be indicated in terms of Nystatin International Unit Equivalents per milliliter (NIUEqs/ml). In other words it may be assumed that that much nystatin as shown by the number of NUIEqs/ml was found in the particular shampoo in place of the actual antifungal agent facilitating easy comparison of their potencies.NIUEqs value of a shampoo is determined as follows. Find out the log nystatin concentration from the standard curve based on In vitro experimental conditions and the actual usage of the shampoos. Further studies are required to match the antifungal potencies determined here with that of the antifungal effects under actual conditions of usage.The outcome of the assay shows that most antidandruff shampoos are effective fungal inhibitors as claimed but there are others with spurious claims. As expected linearity of the nystatin standard curve fell within the narrow range of log transferred nystatin concentrations of 50 to 200 I.U./ml. Four shampoos had inhibition zones corresponding to the standard curve region after diluting 250 times while two others had barely visible zones when tested undiluted. Under the experimental conditions the six shampoos had highly variable potencies 4.07, 4.37, 10227, 10731, 12396 and 18211 NIUEqs/ml in the brands F, E, C, B, D and A, respectively. It may be noted that the potency of nystatin is expressed in International Units (IU) and that of a shampoo expressed in NIUEqs. An obvious difference exists between the A comparatively simple microbiological potency determination method using NIUEqs based on a pharmacopoeial assay procedure has given successful results. The newly proposed antidandruff shampoo potency parameter NIUEqs harmonize the potency determination irrespective of the diversity of the antidandruff agents found in the shampoos. The method is suitable for the screening of diverse antifungal agents that may be found in the shampoos. Zinc pyrithione appear to be the most effective antifungal agent among the samples screened. Shampoos with false antidandruff action claims may be easily detected by the proposed method.


Acarus siro. We detected a second genome-wide significant association with respect to the antigen-specific IgE responsiveness to Tyrophagus putrescentiae at a different locus on chromosome 5. A. siro and T. putrescentiae both belong to the family Acaridae and represent so-called storage or forage mites. These forage mites are discussed as major allergen sources in canine AD. No obvious candidate gene for the regulation of IgE levels is located under the two association signals. Therefore our studies offer a chance of identifying a novel mechanism controlling the host's IgE response.Crosslinking of immunoglobulin E antibodies (IgE) bound at the surface of mast cells and subsequent mediator release is considered the most important trigger for allergic reactions. Therefore, the genetic control of IgE levels is studied in the context of allergic diseases, such as asthma, atopic rhinitis, or atopic dermatitis (AD). We performed genome-wide association studies in 161 Labrador Retrievers with regard to total and allergen-specific immunoglobulin E (IgE) levels. We identified a genome-wide significant association on CFA 5 with the antigen-specific IgE responsiveness to  Immunoglobulin E (IgE) is the class of antibodies that is most frequently recognized for its role in type I hypersensitivity  reactions. In predisposed, atopic individuals IgE is produced against specific common environmental antigens. Most of the IgE is bound on the surface of mast cells through the high affinity IgE receptor (FcεRI). Cross-linking of mast-cell bound IgEs by allergens leads to the release of histamine and many other mediators, and subsequently to allergic reactions in the skin, respiratory tract, or other organs FCER1A) on HSA 1q23 or the IL13, IL4, RAD50 region of HSA 5q31 Total or allergen-specific IgE levels have been analyzed as correlated endophenotypes for different allergic diseases, such as asthma, atopic rhinitis, or atopic dermatitis (AD). IgE levels were analyzed instead of directly using the disease status, as they show a higher heritability than the disease status and are assumed to be less susceptible to confounding environmental factors Dogs are valuable models for many human diseases and the special population structure of purebred dogs greatly facilitates the identification of genetic risk factors Dermatophagoides and especially D. farinae are considered the most important sources of allergen in humans and dogs and several allergens have been identified on a molecular level Tyrophagus putrescentiae, Acarus siro, and Lepidoglyphus destructor. It has been shown that such mites can be present even in un-opened bags of dry dog food. Under suitable environmental conditions up to 90% of commercial dry foods became contaminated with forage mites within a couple of weeks after opening of the bags The major recognized environmental allergens appear to be similar between humans and dogs and include house dust and storage mites, pollens, moulds, and insects We are currently building the LUPA cohort of Labrador Retrievers to gain a better understanding of IgE regulation and its relation to AD. We report here the results of a GWAS with respect to immunological traits in this cohort.Dermatophagoides farinae specific IgG1 and IgG4, total IgE, and 12 additional allergen-specific IgE serum levels against Dermatophagoides farinae, Dermatophagoides pteronyssinus, Tyrophagus putrescentiae, Lepidoglyphus destructor, Acarus siro, Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus, Penicillinum sp, cat epithelium, flea saliva, and Blatella germanica.We collected sera from 161 Labrador Retrievers and determined 15 immunological phenotypes consisting of A. siro is a storage mite considered to be a possible source of antigens that may provoke AD. We determined the A. siro-specific serum IgE levels by an enzyme-linked immunosorbent assay (ELISA) and expressed them in ELISA units (EU). The measured IgE levels ranged from 0 to 3060 EU. The lower reliable detection limit for the A. siro-specific IgE level is 150 EU. We treated the IgE levels as a binary trait and considered dogs with more than 150 EU as IgE-responsive to A. siro and dogs with less than 150 EU as IgE-negative controls.A. siro-specific IgE responders (cases) and 24 controls remained for the final analysis , showed a raw p-value of 2.14×10−9. This is below the Bonferroni-corrected significance threshold of 4.4×10−7. We also determined the empirical significance threshold by performing 100,000 permutations with randomly assigned phenotypes, which yielded a genome-wide corrected p-value of 0.001  that was significantly associated with the IgE response to of 0.001 , Table 1The 27 best associated markers of the GWAS were clustered on CFA 5 in an interval ranging from 79.0–86.7 Mb. Only the 28th best-associated marker resided at a different locus on CFA 37. The observed linkage disequilibrium of the association signals on CFA 5 delineates the associated interval to approximately 3.5 Mb ranging from 79.0–82.5 Mb .IL34), in this interval.The canine genome annotation still remains imperfect. Therefore, we extrapolated the gene content of the associated region from the orthologous human genome interval. The associated region matches to two counterparts on HSA 16, located between 70.1–74.0 Mb and 74.4–74.7 Mb, respectively . This reA. siro-specific IgE levels we also found a significantly associated SNP with respect to the allergen-specific IgE levels against Tyrophagus putrescentiae .It is well known that A. siro IgE-responsiveness is located between 79.0 Mb and 82.5 Mb. In the investigated canine chromosomal region no obvious candidate gene for the IgE-responsiveness is located. The interval contains the IL34 gene encoding a member of the interleukin family, but this gene has so far not been implicated in the regulation of IgE levels IL34 gene has not been associated with IgE levels in human GWAS The locus of the A. siro, but none of the other allergens. This experimental finding is most likely due to the skewed distributions of IgE responders and non-responders with respect to the different antigens investigated. Our detailed analysis of dogs with “A. siro IgE non-responsive” and “A. siro IgE responsive” genotypes at the associated SNP revealed a consistent trend for lower IgE but not lower IgG levels across all antigens in the non-responsive genotype class .A. siro-specific IgE and 25 dogs with low A. siro-specific IgE serum levels (see below). This work was part of a larger effort to build the LUPA AD cohort, which currently consists of 302 Labrador Retrievers. The dogs were clinically diagnosed according to established criteria for canine AD A. siro-specific IgE levels 61 were diagnosed as AD-affected and 75 as controls, whereas among dogs with low levels of A. siro-specific IgE 16 were AD-affected and 9 were controls. The samples were recruited from 2008–2010.We collected 161 serum and EDTA blood samples from Labrador Retrievers, including 136 dogs with high Genomic DNA samples were isolated from EDTA blood with the Nucleon Bacc2 kit . The DNA was genotyped at the Centre National de Génotypage, Evry, France using illumina canine_HD chips containing 174,376 SNP markers. Genotypes were stored in a BC/SNPmax Database version 3.4 (BC/Platforms).Dermatophagoides farinae, Dermatophagoides pteronyssinus, Tyrophagus putrescentiae, Lepidoglyphus destructor, Acarus siro, Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus, Penicillinum sp, cat epithelium, flea saliva, and Blatella germanica were measured by a commercially available test  Allergen-specific serum IgE levels against Total serum IgE levels were also determined by ELISA. Briefly: Plates  were coated with anti-canine IgE monoclonal antibody D9 Dermatophagoides farinae were determined in the sera of AD and control dogs. 96 well plates  were coated with Dermatophagoides farinae extract . After blocking, dog sera were serially twofold diluted and added to the plate. A serum pool from dogs immunized with Dermatophagoides farinae was used to generate a standard curve on each plate. After incubation and washing, monoclonal antibodies specific for canine IgG1 (clone B6) or IgG4 (clone A5) were added to the plates Relative concentration of the IgG subclasses IgG1 and IgG4, specific for Dermatophagoides farinae specific IgG1 and IgG4 as well as the total IgE serum levels were transformed into categorical traits by using the median as cut off.The Dermatophagoides farinae specific immunoglobulin levels , we removed 13 dogs that had received allergen-specific immunotherapy with Dermatophagoides farinae extracts. We also removed markers with minor allele frequency (MAF)<5% and markers strongly deviating from Hardy-Weinberg equilibrium (p<10−5). We performed an allelic association study using the h2a2 function, which uses a mixed-model approach, which uses the kinship matrix estimated from the marker data to correct for population stratification. Significance levels were determined by the Bonferroni correction (pBonf=0.05/number of markers). Additionally, empirical significance thresholds were also determined by performing 100,000 permutations of the dataset with arbitrarily assigned phenotypes.We used GenABEL We used the dog build 2 and the human build 37 assemblies for all analyses. We used BLASTN searches to define the orthologous human chromosomal regions corresponding to the associated interval on CFA 5. For the candidate gene inspection we used the human annotation provided by NCBI (build 37.3).Figure S1Multidimensional scaling (MDS) plot showing the genomic kinships between the analyzed Labrador Retrievers. This plot visualizes the overall genetic distances between the dogs based on 2,000 markers randomly selected out of the total of 113,021 SNP markers. Cases and controls do not form separate clusters, which is an essential prerequisite for a successful GWAS.(TIF)Click here for additional data file.