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J. Biol. Chem.281, 4663-4670.
Apoptotic cells, at all stages of the death process, trigger characteristic signaling events that are divergent from and dominant over those triggered by necrotic cells.2006

Notes: This study investigates the role of the ERK1/ERK2 (MAPK) pathway in growth regulation of venous ulcer fibroblasts. Human normal (n-fb) and venous ulcer (w-fb) fibroblasts were isolated and grown in culture in the presence or absence of the MEK-1 inhibitor PD 98059. AntiACTIVE® MAPK pAb was used in immunoblots to determine the expression of phosphorylated ERK1/ERK2 in n-fb and w-fb. In separate experiments, human neonatal foreskin fibroblasts were obtained and cultured in the presence of wound fluid from venous ulcers plus or minus PD 98059 to determine whether or not wound fluid had an inhibitory effect on expression of MAPK ERK1/2. Immunoblot analysis using AntiACTIVE® MAPK pAb indicated that wound fluid decreased phosphorylated ERK1/ERK2 expression; addition of PD 98059 did not further decrease expression of phosphorylated ERK1/ERK2 in these cells. (3537)

Notes: Serum-starved PC3 and LNCaP prostate cancer cells were treated with various concentrations of ghrelin over a time course. Western blots of cell lysates were probed with Anti-ACTIVE® JNK pAb, Anti-ACTIVE® p38 pAb, Anti-ACTIVE® MAPK pAb and Anti-ERK(1/2) pAb to assess activation of MAPK pathways. (3434)

Notes: The authors of this study sought to determine whether both MAP kinase and Cdk1/cyclin B kinase are required for mitotic entry and calcium signals in early sea urchin embryos. Treating embryos with MEK Inhibitor U0126 12 minutes after fertilization inhibited MAP kinase activity in the early embryos. Anti-ACTIVE® MAPK pAb was used for immunoprecipitation of active MAP kinase at various times after fertilization. (3506)

Notes: The Anti-ACTIVE® MAPK polyclonal antibody was used to immunohistochemically stain and type patient ovarian serous carcinomas. The researchers used a 1:500 dilution of the antibody on paraffin fixed tissue sections on tissue microarrays. A secondary peroxidase staining kit was used to complete the immunohistochemical staining. In these studies, the ovarian serous carcinomas displayed increased MAPK expression and activity. Western blots were also performed on tissue lysates using a 1:3000 dilution of the antibody. (3210)

Notes: In this paper, the authors describe use of ImProm-II™ Reverse Transcriptase to transcribe cDNAs for Quantitative RT-PCR. Reverse transcription (RT) reactions were performed with 1μg total RNA from treated rat PC12 cells. Light Cycler reactions were setup with 1-3μl cDNA from the RT reactions. The researchers incubated cells with EGF and bFGF and analyzed HIF-2α mRNA levels. For these experiments, PC12 cells were incubated with 30ng/ml EGF or 50μM bFGF for 8 hours. Erk1&2 phosphorylation was examined by Western blot using the Anti-ACTIVE® MAPK pAb. Western results were visualized by chemiluminesent detection and analyzed with a digital luminescent image analyzer. The Dual-Luciferase® Reporter Assay System was used to analyze PC12 cell-expressed luciferase from pEpoEm1-luc and pEpoE-luc promoter constructs that were normalized with the pRL-TK vector. Cells were harvested 17-18 hours post transfection and treatment. (2725)

Notes: This paper describes a study of the effect of endogenous neurotrophins on cortical neuron development. Cortical progenitor cells were isolated from mouse embryos and cultured. These precursor cells express both BDNF and NT-3 as well as the corresponding TrkB and TrkC receptors. BDNF and NT-3 signal via Trk receptors to activate the PI3-kinase and MEK pathways. These pathways serve distinct functions, PI3-kinase being essential for progenitor survival and MEK for the differentiation of neurons but not glial cells. The progenitor cell cultures were treated with either Anti-Human BDNF pAb or Anti-Human NT-3 pAb at 20μg/ml to block the function of the endogenous neurotrophins. Anti-ACTIVE® MAPK pAb was used to assess levels of activated MAPK by Western blotting of cell extracts. (2778)

Notes: The role of integrin alpha(IIb)beta(3) in focal adhesion kinase activation and MAPK signaling was studied using Chinese hamster ovary and human kidney 293 cell lines expressing either the Leu(33) or Pro(33) isoform of beta(3). Compared with Leu(33) cells, Pro(33) cells demonstrated substantially greater activation of ERK2 (but not MAPK family members JNK and p38) upon adhesion to immobilized fibrinogen (but not fibronectin), and upon integrin cross-linking. ERK2 activation was mediated through MAPK kinase and required phosphoinositide 3-kinase signaling and an intact actin cytoskeleton. Levels of activated MAPK family members ERK1 and 2, JNK, and p38 were assessed by western blotting using Anti-ACTIVE® MAPK (1:5000 dilution), Anti-ACTIVE® JNK (1:5000 dilution), and Anti-ACTIVE® p38 (1:1000 dilution) pAbs. Anti-ERK 1/2 pAb was used at a 1:5000 dilution as a control for total protein amounts loaded on the blots. (2789)

Notes: This paper investigates the mechanism of estrogen receptor alpha (ERalpha) degradation. Loss of ER is associated with aggressive breast tumors and poor clinical outcome. ERK7 is shown to be a specific regulator of ERalpha degradation in human breast cells. Several members of the MAPK family were examined to determine if they regulate ERK7 turnover. Levels of activated MAPK were examined by Western blotting of extracts of BHK cells that had been co-transfected with different kinases. The Anti-ACTIVE® MAPK pAb was used to detect dually phosphorylated ERK p42/p44 in these lysates. Overexpression of ERK7 was shown to specifically enhance degradation of ERalpha in BHK cells. ERK7 and active MAPK levels were assessed in breast cancer cell lines as well as normal and cancerous human breast tissue. Lowered ERK7 levels correlated with ERalpha-positive tumors, suggesting that loss of ERK7 leads to higher levels of ERalpha in these cancers. (2769)

Notes: This paper investigates the pathway by which follicle stimulating hormone (FSH) activates ERKs (MAPK) p42 and p44 in rat primary granulosa cells. The phosphorylation state of MAPK p42/p44 was assessed by Western blotting using the Anti-ACTIVE® MAPK pAb. Phosphorylated MAPKs were localized to the nucleus of granulosa cells by immunocytochemistry using Anti-ACTIVE® MAPK pAb. Cells were stimulated, fixed with 3.7% formaldehyde, permeabilized with 1% Triton X-100 in PBS, washed, blocked for 1 hour in 1% bovine serum albumin in PBS and incubated overnight at 4°C with a 1:100 dilution of the antibody in PBS containing 1% bovine serum albumin. (2768)

Notes: Esophageal contraction has been shown to be protein kinase C (PKC) dependent. These authors examined the involvement of ERK1/ERK2 and p38 MAPKs in PKC-dependent esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) contraction. Feline ESO and LES circular smooth muscle was homogenized and the level of phosphorylated MAPK was studied by Western blotting using the Anti-ACTIVE MAPK® pAb. (2771)

Notes: The solution structure of PAC-1, a MAPK phosphatase, was determined in this study. An activated and dually phosphorylated ERK was purified by expressing a human cDNA in E. coli BL21(DE3) cells co-expressing a constitutively active MEK1. The phosphorylation state of this purified ERK was determined by Western blotting with Anti-ACTIVE MAPK pAb, Rabbit, (pTEpY), Anti-Phosphotyrosine pAb, and Anti-pT183 MAPK pAb. The Western blots were detected with Donkey Anti-Rabbit IgG (H+L), AP. (2689)

Notes: Fibroblast growth factor (FGF) signaling is necessary for proliferation and differentiation of chicken lens cells. The transcription factor L-Maf is a lens differentiation factor that appears to mediate FGF signaling. This paper shows that L-Maf is repressed by FGF/ERK signaling and that L-Maf is phosphorylated by ERK. Both Anti-ACTIVE MAPK® pAb and Anti-ERK1/2 pAb were used to determine MAPK levels by Western blotting. Anti-ACTIVE® MAPK pAb was also used in immunocytochemistry analysis of cultured chick lens cells. (2773)

Notes: The transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) occurs in cells as the transcriptional activator liver-enriched activating protein (LAP) and, in truncated form, as liver-enriched inhibitory protein (LIP), which inhibits transcription. Previous studies have shown that growth hormone (GH) promotes dephosphorylation of both LAP and LIP. This study investigated phosphorylation states of LAP and LIP by isoelectric focusing (IEF). IEF revealed that GH not only promotes dephosphorylation, but also promotes rapid and transient phosphorylation of murine C/EBPbeta on Thr188 in mLAP and Thr37 in mLIP, sites that correspond to the conserved mitogen-activated protein kinase (MAPK) consensus sequence. Western blotting of 3T3-F442A cell lysates showed a correlation between activation of MAPK (ERK1/2) and phosphorylation of C/EBPbeta. Active MAPK was measured using the Anti-ACTIVE MAPK® pAb, and total ERK levels were measured by Western blotting with Anti-ERK1/2 pAb. (2774)

Notes: The authors investigated the relationship between signaling events at the synapse and cascades that translate the signal to the nucleus. Rat hippocampal slices were prepared and maintained in a perfusion chamber and stimulated with electrodes. The same slices were then stained for phosphorylated ERK using Promega's Anti-ACTIVE® MAPK pAb. (2437)

Notes: The authors studied the signaling cascades activated by Toll-like receptor 2 (TLR-2) and TLR-4, transmembrane proteins that respond to a variety of microbial derived molecules and signals such as lipopolysaccharide (LPS), lipid A among others. Primary C3H/OuJ macrophages were treated with E. coli K235 LPS or Pam3Cys for 30 minutes and total cell lysates were prepared for immunoblot analysis. MAPK phosphorylation was measured using Promega's Anti-ACTIVE® MAPK pAb. (2430)

Notes: Harpin proteins are a group of effector proteins involved in plant pathogenesis of several phytopathogenic bacteria. The role of these proteins in pathogenesis was examined and the authors found that MAPK activation is required. Activated MAPK was detected by Western blot analysis with an antibody raised specifically against the dually phosphorylated (active) form of MAPK. Thirty micrograms of tobacco protein extract was separated by SDS-PAGE, transferred to nitrocellulose membrane, and blotted with the Anti-ACTIVE® MAPK pAb. A secondary goat anti-rabbit IgG antibody coupled to alkaline phosphatase was used to visualize the immunoreactive proteins. (2397)

Notes: The transcriptional targets of the MAPK pathway in a TGFβ1 induced model of epithelial-mesenchymal transitions are explored. Activation of MAPK signaling regulated genes with functions in cell-matrix adhesion and endocytosis. Human keratinocytes were cultured in the absence or presence of 15µM U0126. To monitor MAPK activation and inhibition, the Anti-ACTIVE® MAPK pAb was used in Western blots to detect the dually phosphorylated form of MAPK. Results were normalized to total level of active and inactive MAPK by Western blot analysis with the Anti-ERK 1/2 pAb, Rabbit. (2389)

Notes: The function of Integrin Linked Kinase (ILK) in cell interactions with extracellular matrix was studied. ILK activation is an important event in the integrin-mediated signal pathway and is necessary for neurite outgrowth in serum-starved mouse N1E-115 neuronal cells on laminin. Activation of p38 MAP kinase is necessary for the ILK-mediated signal leading to integrin-dependent neurite outgrowth. Activation of MEK and ERK does not appear to be involved in this process. Both Anti-ACTIVE® MAPK pAb and Anti-ERK1/2 pAb were used to assess levels of active and total p42/p44 in cell extracts by Western blotting. (2770)

Notes: A possible linkage between glutamate signalling and the MAPK cascade in rat primary cortical astrocytes was examined. Levels of activated, dually phosphorylated MAPK were determined using the Anti-ACTIVE® MAPK pAb whereas total MAPK levels were monitored with the Anti-ERK 1/2 pAb, Rabbit. The MEK inhibitor U0126 at 1µm completely abolished ERK phosphorylation in response to glutamate release. Following SDS-PAGE, total protein was transferred to a PVDF membrane and blotted with a 1:5000 dilution of the Anti-ERK 1/2 pAb, Rabbit or a 1:10000 dilution of the Anti-ACTIVE® MAPK pAb overnight at 4°C. (2390)

Notes: The role of JNK, p38 and MAPK signaling pathways in delayed neuronal death after transient forebrain ischemia was examined using Western blotting and immunohistochemistry. Gerbil hippocampi were homogenized, subjected to SDS-PAGE, blotted to PVDF membranes and incubated with one of the following primary antibodies: Anti-ACTIVE® JNK pAb (1:2000 dilution), Anti-ACTIVE® p38 pAb (1:1000 dilution), and the Anti-ACTIVE® MAPK pAb (1:2000 dilution).The secondary antibody was the Donkey Anti-Rabbit IgG (H+L), AP, diluted 1:10,000 for JNK and p38 and 1:5000 for ERK. For immunohistochemistry, gerbils were perfused with 4% paraformaldehyde. Frozen 40µm sections were stained with either the Anti-ACTIVE® JNK pAb and the Anti-ACTIVE® p38 pAb. (2402)

Notes: Gerbils were treated with 3-nitropropionic acid intraperitoneally for 1 to 4 days. Sections of the CA1 area of the hippocampus were analyzed for activation of various mitogen activated kinases. The c-Jun N-terminal kinase was activated by the treatment as judged by immunohistochemistry of 40µm frozen sections with the Anti-ACTIVE® JNK pAb. (0313)

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