Protocols for measuring phosphorylation, subcellular localization, and kinase activity of Hippo pathway components YAP and LATS in cultured cells

Summary The Hippo pathway plays critical roles in cell growth, differentiation, development, tissue homeostasis, and cancer. Here, we describe protocols to measure phosphorylation, subcellular localization, and kinase activity for core Hippo pathway components YAP and LATS. The phos-tag technology is particularly useful to quantitatively detect protein phosphorylation even without a phosphospecific antibody. We use HEK293A cells as an example, but the protocols can be applied to other cell lines and tissues. For complete details on the use and execution of this profile, please refer to Luo et al. (2020).


SUMMARY
The Hippo pathway plays critical roles in cell growth, differentiation, development, tissue homeostasis, and cancer. Here, we describe protocols to measure phosphorylation, subcellular localization, and kinase activity for core Hippo pathway components YAP and LATS. The phos-tag technology is particularly useful to quantitatively detect protein phosphorylation even without a phosphospecific antibody. We use HEK293A cells as an example, but the protocols can be applied to other cell lines and tissues. For complete details on the use and execution of this profile, please refer to Luo et al. (2020).

BEFORE YOU BEGIN
Note: For detection of YAP phosphorylation (Part I), follow steps 1-4 in BYB and steps 1-12 in Step by Step; For detection of LATS kinase activity (Part II), follow steps 1-2 of BYB and steps 13-23 in Step by Step; For detection of YAP or TEAD subcellular localization (Part III), follow steps 1-2 of BYB and 24-36 in Step by Step.

Prepare materials and cells
Timing: 3 days 1. Start a new HEK293A cell culture. Resuscitate low passage HEK293A cells and seed into a 100 mm Cell Culture Dish. Grow cells in a standard culture incubator and passage the cells at the log phase to always keep cells healthy and > 90% viable.
Note: Low passage cells are used to ensure cell quality. It is not recommended to use cells with altered morphology or growth rate, or cells that have been passaged less than 2 times or too many times from frozen stock.
Note: Cells should be tested for and free of mycoplasma contamination. Unlike bacterial or fungal contaminations, mycoplasma infection cannot be detected by visual inspection and may not noticeably affect cell growth. However, mycoplasma contaminations have been reported to alter DNA, RNA and protein synthesis (Drexler and Uphoff, 2002). Short tandem repeat (STR) analysis should also be performed to ensure the quality and integrity of human cell lines.
Note: All cell culture plates or dishes are incubated at 37 C with humidified 5% CO 2 in a standard culture incubator (Thermo Fisher Scientific).
2. Prepare DMEM with 10% fetal bovine serum (FBS) and 13 antibiotic solution for cell culture of HEK293A beforehand. The cell culture medium are stored at 4 C and warmed to 37 C before use. Ensure there are sufficient solutions and reagents needed for this protocol. Especially 43 SDS-PAGE Sample Buffer, 103 SDS Running Buffer and 103 Transfer Buffer (Methanol-free) need to be prepared in advance.
CRITICAL: When preparing the above various buffers, high purity compounds within the validity period are strongly recommended, such as AR grade Tris base, glycine and SDS, etc. Expired or impure compounds will affect the results of the Phos-tag gel. For example, if Tris-HCl buffer is stored at 15 C-25 C for more than a year, white flocculent precipitation will occur in the buffer and affect the pH of the buffer.
Note: It is better to prepare fresh blocking buffer (5% milk) and antibody diluent (5% BSA) since the milk and BSA in the buffer will support microbe growth, which can have an adverse impact on the results of the immunoblot analysis.
3. Because a lot of heat will be generated during the transfer process, the 13 transfer buffer (containing methanol) must be pre-cooled at 4 C at least 24 h in advance. 4. The formula for the preparation of 6% Phos-tag gels or 8% Regular gels is listed in the materials and equipment. a. For separating gels: 10 mL reaction mixture for two gels. i. Try to pour ddH 2 O into the two glass sheets' gaps firstly to check if there is any leakage.
ii. Add 10% APS (ammonium persulfate) and TEMED last. After mixing, separating gel mixture should be carefully added between the two glass sheets. iii. Add 1 mL of isopropanol at the top of the gel mixture to ensure uniform gel polymerization. b. For stacking gel: 4 mL reaction mixture for two gels.
i. The comb (15-well, 1.0 mm) should be gently inserted to avoid bubbles following the addition of the stacking gel solution. ii. After polymerization, the comb should be carefully pulled out to avoid bending of the gel stripes between lanes. Store at 4 C for up to 3 months. Pre-warm to 37 C before use. Store at 4 C for up to 6 months. Take out just before use and keep on the ice.

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CRITICAL: Reagents used may have potential hazards, such as acrylamide, SDS, beta-mercaptoethanol, TEMED, methanol and paraformaldehyde. Their harmful or toxic include one or more of the following: skin/eye corrosion/irritation, allergic skin reaction, genetic defects, carcinogenicity, etc. When handling these reagents, preventive measures include but are not limited to protective gloves/protective clothing/eye protection/face protection. Experiments should be performed in a well-ventilated area, and wash face, hands and any exposed skin thoroughly after handling.
Alternatives: In addition to Cell Signaling Technology, some Hippo pathway antibodies can also be purchased from the following companies, such as Santa Cruz Biotechnology, Abcam, and Bethyl Laboratories. However, we recommend using the antibodies from the sources listed in the reagent table because their quality has been verified through many experiments.

STEP-BY-STEP METHOD DETAILS Part I: Detection of YAP phosphorylation
Note: This protocol is modified from Chen et al. (2019).

Seeding HEK293A cells
Timing: 1 h In this section, all cell culture steps are to be performed under ''aseptic conditions'' in a cell culture hood.
1. Once HEK293A cells reach 80%-90% confluency, they are ready for passage. a. Aspirate old culture media from the 100 cm culture dish and gently rinse the cells with $3.0 mL of sterile 13 PBS. b. Add 2 mL of pre-warmed 0.25% Trypsin-EDTA directly to the cells and incubate at 37 C for 1-2 min until cells become shrunk and round and have detached from the bottom of the dish. During the digestion period, you can tap the edge of the culture dish with your hand. c. Add 3 mL of pre-warmed DMEM with FBS to neutralize Trypsin-EDTA and mix the cells by pipetting up and down repeatedly until achieving a single-cell suspension, and then transfer the approximately 5 mL cell suspension to a 15 mL conical centrifuge tube. d. Centrifuge the tube at 3003g for 3 min, discard the supernatant and resuspend the cell pellet with an appropriate amount of pre-warmed DMEM with FBS and mix well by pipetting up and down several times. e. Count cells using the automated cell counter (Countstar) and plate the cells into 6-well plates at a density of 5310 5 to 8 310 5 cells/well, depending on the experimental needs. Add prewarmed DMEM with FBS to make a final volume of 2 mL/well. Culture the cells in a standard incubator for 24 h.
Note: The amount of cells can be scaled down to 12 or 24 well plates to meet experimental needs.
CRITICAL: Cell density will affect the phosphorylation of Hippo pathway components, including YAP, TAZ, LATS and MST (Meng et al., 2016). Seed appropriate cell numbers into each well to ensure that the cells reach the desired confluence the next day.
CRITICAL: It is recommended to overexpress MST1 by transient transfection because the phosphorylation signal of endogenous MST1 in HEK293A cells is a little weak and difficult to be detected.

Collect cell lysis for immunoblot analysis
Timing: 0.5 h for collect cell lysate, 2 days for immunoblot analysis In this section, the cell lysate will be subjected to immunoblot analysis to detect the phosphorylation for Hippo pathway components. This experiment is described separately according to the two different types of gels, i.e., Phos-tag gel for YAP phosphorylation analyses and regular gel for pYAP(S127), pLATS1 (T1079) and pMST1(T183) analyses.
2. Collect cell lysate: Aspirate old culture media as completely as possible. Then harvest cells by directly adding 300-1000 mL 13 SDS-PAGE Sample Buffer per well in 6-well plates, and shake the plate thoroughly using a plate shaker at 1503g for at least 10 min. Transfer the sample to 1.5 mL microcentrifuge tubes and heat at 95 C-101 C for 5 min. Centrifuge at 16,0003g for 5 min at 15 C-25 C and store at À20 C for subsequent immunoblot analysis.
Note: You can treat the cells with designed stimulation before collecting the cells, such as heat shock, serum starvation, small molecule compounds, osmotic stress, etc.
Note: There is no need to add Protease/Phosphatase Inhibitor Cocktail to the sample buffer since all protein is denatured quickly. Cell lysates prepared in the 13 SDS-PAGE Sample Buffer can be stored at À20 C for three months.
Note: No need to determine the protein concentrations using a commercial Bradford assay or BCA Protein Assay Kit as long as each culture well has similar amount of cells. Their concentrations in different samples can be normalized by adjusting the volume of the Sample Buffer.
In general, about 100 mL of Sample Buffer is used for every 2 3 10 5 cells in the well. For ll OPEN ACCESS example, when the HEK293A cells reach 100% confluence in a 6-well plate, the number of cells is approximately 1.5 3 10 6 and 800 mL of Sample Buffer should be added. If the cell confluence only reaches 50% in the treatment well, add only 400 mL of Sample Buffer accordingly.
3. Loading sample: Take out the comb carefully and aspirate any remaining liquid in the wells before loading the samples. Load 10-15 mL of sample per well.
Note: Protein ladder should be loaded on the leftmost and rightmost side of the sample well, and judge your sample loading order by the color shade of protein ladder. Dilute the prestained protein ladder from Thermo Fisher Scientific using 13 SDS-PAGE Sample Buffer (250 ml protein ladder diluted in 1 ml Sample Buffer), and load the same volume of blank sample buffer to any empty wells to ensure uniform electrophoresis.
4. Electrophoresis: Start electrophoresis at 80 V for 30 min, until all the samples enter the separating gel.
For Phos-tag gel, then run at 100 V for about 2.5 h until the 55 kDa marker in the pre-stained protein ladder is around 1.5 cm from the bottom of the gel (YAP protein is above the 70 kDa marker).
For Regular gel, then run at 120 V for about 1.5 h until the bromophenol blue indicator line is around 0.5 cm from the bottom of the gel.
5. Transferring: The PVDF membrane with a 0.45 mm pore size is activated by soaking in methanol for 2 min. Assemble the wet transferring system, and transfer for 2 h (Phos-tag gel) or 1 h (Regular gel) at 4 C in the cold room or refrigerator. Set constant current at 370 mA for one Trans-Blot tank, or 740 mA for two Trans-Blot tanks.
Note: The filter paper used for blotting transfer should be chromatography paper. The thickness of the filter paper on one side of Gel Holder Cassettes should be 0.7-0.8mm. Therefore, use 2 sheets of filter paper with a thickness of 0.4 mm on one side. If use commercially available filter paper with a thickness of 0.8 mm, one sheet on each side is sufficient.
CRITICAL: It takes more time to transfer YAP protein from phos-tag gel to PVDF membrane than that of regular gel. Therefore, in order to avoid overheating of the transfer buffer, it is strongly recommended to change the Ice Cooling Unit after 1 h's phos-tag gel transferring.
6. Wash the whole PVDF membrane one time with 13 TBST for 5 min. 7. Block with 5% milk in TBST for 1 h at 15 C-25 C with gentle rocking. 8. Dilute the primary antibody in 5% BSA in TBST. Incubate and rock the membrane with primary antibody around 10-15 h at 4 C.
Note: The primary antibody should be diluted in 5% BSA at concentrations recommended by the manufacturer. Details as follows: YAP, 1:1,000; LATS1, 1:2000; MST1, 1:2000; pYAP (S127), 1:1000; pLTAS1 (T1079), 1:1000; pMST1 (T183), 1:500; GAPDH, 1:4000. The primary antibody solution can be used multiple times depending on the quality of the antibody. For a short term (within a week), the antibody solution can be stored at 4 C to avoid freeze-thaw cycles. For a long time (more than a week), we suggest to store the antibody solution at À20 C.
CRITICAL: The antibody against YAP protein is used as primary antibody in the Phos-tag gel analysis. Please do not use the phosphoantibodies of pYAP(S127).

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STAR Protocols 3, 101102, March 18, 2022 9. The next morning, pipette out the primary antibody and save for future use (the antibody can be reused for 5 times usually). Wash the blot 3 times with 13 TBST with rocking for 10 min each. 10. Incubate with HRP-linked secondary antibody (diluted in 5% milk in TBST at 1:5000) for 1 h at 15 C-25 C.
Note: Be sure to use a secondary antibody that can recognize the source of the specific primary antibody (most are from rabbit or mouse).
11. Wash the blot 4 times with 13 TBST for 15 min each with rocking. 12. Incubate the membrane with the Western HRP substrate for 1 min at 15 C-25 C. Remove the membrane from the working reagent and absorb any excess liquid or bubbles with Kimwipe or filter paper, place the membrane between two transparent plastic sheets or wrap the membrane with Saran wrap, expose to autoradiography film in an X-ray film cassette, develop the film with a film developer in a dark room.
Note: The exposure time varies for different target proteins and antibodies. A good starting point is an exposure time of 1 min. Avoid over exposure time, which will saturate the signal band and conceal the changing trend of the target proteins.
Note: This protocol certainly does not only apply to 6 well plates. To ensure that sufficient amount of LATS1 is recovered for subsequent IP experiments, it can be scaled up in 100 mm culture dishes to meet experimental needs.
CRITICAL: Cell density will affect the Hippo pathway, including the localization of YAP and activity of LATS1 kinase (Zhao et al., 2007). Seed appropriate cell numbers into 6 well plates or dishes to make sure that the cells reach the desired confluence next day.
Alternatives: 24 h after seeding the cells, expression plasmids or siRNAs transfection may be performed if the expression of a gene needs to be modulated. The transfected HEK293A cells can be reseeded.

Cell lysis, LATS immunoprecipitation and kinase assay
Timing: around 5 h 14. Prewash the Magnetic Beads: Thoroughly vortex (max speed, 30 s) the commercial magnetic beads tube, then pipette an appropriate amount of beads (10 mL/Sample) into a new 1.5 mL microcentrifuge tube, wash the beads 3 times with mild lysis buffer (MLB) and final resuspend with MLB with protease/phosphatase inhibitor cocktails (20 mL/Sample).
Note: Consider the amount of loss during operation, prepare the magnetic beads for (n + 2) samples.
Note: Detailed wash method: add 1 mL of wash buffer to the tube containing an appropriate amount of beads, vortex thoroughly and place the tube into a magnetic stand for 2 min to ll OPEN ACCESS collect the beads against the side of the tube; remove the supernatant; take out the tube from the magnetic stand, repeated the above washing for total 3 times.
15. For HEK293A cells in the 6 well plates or dishes, gently aspirate old media, wash twice with cold PBS gently.
Note: You can treat the cells with various signals before collecting the cells, such as heat shock, serum starvation, small molecule compounds, osmotic stress.
CRITICAL: HEK293A cells detach from the surface of plate very easily. Thus, all rinsing steps should be performed carefully and gently, as to not cause HEK293A detachment. LATS activity can be rapidly perturbed by many environmental factors, thus ice-cold PBS should be used to wash the cells and harvest should be completed promptly on ice.
16. Add 1 mL MLB with protease/phosphatase inhibitor cocktails directly onto the cells for each sample, shake the plates or dishes at 4 C for 10 min, and then transfer the cell-containing solution to 1.5 mL microcentrifuge tubes. 17. Spin at 16,0003g, 4 C for 10 min, to separate all the cell debris from the supernatant, and transfer the lysis supernatant to a new 1.5 mL microcentrifuge tubes, which need to be maintained on ice. 18. Add LATS1 antibody (CST, 1: 1000) and pre-washed magnetic beads resuspended in MLB (20 uL/sample) (from step 14) together into the lysis samples prepared in step 17, invert gently to mix at 4 C for 2 h. Include a negative control with the normal rabbit IgG (the LATS1 antibody is rabbit IgG). Photos of sample tubes with an inverted mixing at 4 C can be seen in Figure 1A.
CRITICAL: In conventional IP experiments, after adding specific antibodies, the samples usually be incubated around 10-15 h at 4 C with mixing. But in the kinase assay experiment, to ensure the activity of LATS1 kinase, the incubation time should not exceed 2 h.
Note: The concentration of Normal Rabbit IgG is 1mg/mL, which is higher than the concentration of LATS1 antibodies from the CST company (around 0.2 mg/mL). Thus, the IgG control needs to be diluted with MLB to the same concentration before use.
19. After 2 h's incubation, collect the beads with a magnetic stand, remove the supernatant, then wash 5 times with MLB and subsequently 1 time with 13 Protein Kinases Buffer. A photo of sample tubes on a magnetic stand to collect the beads against the side of the tube is shown in Figure 1B. CRITICAL: The remaining reagent components in the tube may affect the kinase reaction, so it must be thoroughly washed with MLB at least 5 times, and the 13 Protein Kinases Buffer should be used for the last washing.
Note: Detailed wash method: add 1 mL of wash buffer into each tube, invert the tube several times and gently shake at 4 C for 5 min, then place the tube back into a magnetic stand for 2 min to collect the beads against the side of the tube; remove the supernatant; repeat above washing steps.
20. Add 40 mL of kinase reaction buffer with substrate GST-YAP per sample directly to the abovewashed beads in step 19, mix gently by finger tap, and incubate on a heat shaker with 300 rpm at 30 C for 30 min.
CRITICAL: To ensure the reaction quality, it's better to make the reaction buffer mix just before use and samples can be manually mixed by a finger tap every 10 minutes during the incubation.

21.
After 30 min at 30 C, add 13.3 mL of 43 SDS-PAGE sample buffer to the tube, vortex to mix completely and then heat the samples at 100 C for 5 min. 22. Spin down the magnetic beads/samples at 16,0003g, 15 C-25 C for 10 min, then magnetically separate the beads and transfer the supernatant to a new microcentrifuge tube and saved at À20 C for immunoblot analysis.
Pause Point: Lysates prepared for immunoblot analysis can be stored at À20 C for several weeks.

Detection of YAP phosphorylation using immunoblot analysis
Timing: 2 days 23. Please refer to the corresponding step 2-12 in Part I.

Part III: detection of YAP or TEAD subcellular localization
Note: This protocol is modified from Lin et al. (2017).
For most information, please refer to the corresponding steps in "Part I". For the last step, plate cells into 12-well plates on sterile round coverslips at a density of 2 3 10 5 cells/1 mL/well. Culture the cells in a standard incubator around 10-15 h.
Note: For Immunofluorescence assay, usually a 24-well plate or a 12-well plate is sufficient to meet experimental needs, and it is not necessary to use a 6-well plate. When seeding the cells into the well, make sure no air bubbles are trapped underneath the coverslips and get an even distribution of cells.
Note: The coverslips need to be sterilized by immersing in ethanol or autoclaved. If using ethanol for sterilization, the coverslips need to dry completely by evaporation before use.

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CRITICAL: Cell density will affect the subcellular localization of YAP/TAZ or TEAD (Meng et al., 2016). Seed appropriate cell numbers into 12 or 24 well plates to make sure that the cells reach the desired confluence the next day.
Alternatives: To enhance cell adherence, coverslips may be pre-coated with poly-L-ornithine as follows: a. Use sterile forceps to carefully place one sterile coverslip into one well of the 12-well plates. b. Pretreat the coverslip with poly-L-ornithine solution diluted 1:20 at 37 C for 20 min. For one well, dilute 25 mL poly-L-ornithine (0.1 mg/mL) into 500 mL sterile 13 PBS (Final working concentration 0.005 mg/mL). c. Aspirate the coating solution and wash with PBS for several times. Keep the plates in a tissue culture hood for a period of time to allow the coverslip to dry completely. d. Then seed cells on poly-L -ornithine-coated coverslips.
CRITICAL: HEK293A cells are very easy to fall off the coverslip during the permeabilization and wash steps of immunofluorescence. It is strongly recommended to coat the coverslips.

Immunofluorescence staining
Timing: 2 days 25. Aspirate the old medium in wells, rinse them couple times with PBS.
Note: You can treat the cells with various signals before staining, such as heat shock, serum starvation, small molecule compounds, osmotic stress.
Note: The goal of fixation is to maintain the cellular structure as close as possible to the native state. There are several fixation methods suitable for IF, like paraformaldehyde or methanol. You can choose the best one for your target proteins.
27. Wash with PBS 3 times. 28. Permeabilization with 500 mL 0.2% Triton X-100 in PBS for 10 min at 15 C-25 C. Note: For double/triple staining, the primary antibodies can be mixed before being applied to the coverslips, as long as they can be recognized by different secondary antibodies (such as mouse vs rabbit).
32. Remove the primary antibody, and wash with PBS 3 times. 33. Incubate samples with the Goat anti-Mouse IgG or Goat anti-Rabbit IgG second antibody prepared in 2% BSA in PBS (1:1000 dilution) at 15 C-25 C in the dark for 1 h.
Note: Use high-quality and cross-adsorbed the Alexa Fluor secondary antibody conjugates.
34. Wash with PBS 3 times. 35. Mount the coverslips at the center of the slide with ProLongâ Gold Antifade Reagent with DAPI, and wait for several hours to let the mounting medium solidifies. Draw off excess mountant before they are examined by microscope.
Alternatives: The concentration of DAPI that comes with the commercial mountant is very low, resulting in weak DNA staining. One can stain the samples with 1-10 mg/mL DAPI working solution at 15 C-25 C in the dark for 5 minutes before mounting. The DAPI stock solution is usually 1 mg/mL prepared in pure water.
CRITICAL: Because the fluorescence intensity of the sample is easily bleached during confocal photography, a high-quality antifading mountant should be used. Make sure the coverslip with the cell-side is down. Never try to move the coverslip once it falls on the slide or try to squeeze the few air bubbles trapped between the coverslip and the slide, which will damage the structure of the cells.
36. Images were captured with Olympus FV1000 confocal microscopy or microscope available to you. Immunofluorescence data were collected by NIS-Elements Advanced Research 5.11.00 imaging software and the signals from the channels were merged using ImageJ (1.52a) software.

EXPECTED OUTCOMES
Phos-tag is a compound that bind phosphate in the presence of Mn 2+ . Phos-tag was developed to detect protein phosphorylation (Kinoshita et al., 2009). The migration rate will be slower for proteins with a higher degree of phosphorylation in Phos-tag gels. It has been well established that there are at least five LATS1 phosphorylation sites in YAP protein (Zhao et al., 2007). Thus, in Phos-tag gel, YAP can be resolved into multiple bands between 70 kDa to 100 kDa, representing YAP with different levels of phosphorylation ( Figure 2A). Latrunculin B (LatB) induces actin depolymerization and activates the Hippo pathway. The top band (around 100 kDa) represents YAP with the highest degree of phosphorylation which has the slowest migration. The bottom band represents non-phosphorylated YAP (around 70 kDa), which has the fastest migration. Conversely, in regular gels the phosphorylated forms are poorly separated from the unphosphorylated form. Therefore, antibodies for pYAP (S127), pLTAS1 (T1079) and pMST1(T183), essentially show a single band for each protein ( Figure 2B).
The activity of LATS1 kinase is positively correlated with the degree of YAP phosphorylation if substrate is not limiting (Meng et al., 2016). Thus, we usually directly ascertain LATS1 activity using glutathione S-transferase (GST)-YAP as a substrate. LATS1 immunoprecipitated from heat-shocked HEK293A cells exhibited low activity to phosphorylate YAP on S127 (Figure3) (Luo et al., 2020).
Once the Hippo pathway is activated, the LATS-dependent phosphorylation of YAP/TAZ results in 14-3-3 binding and cytoplasmic localization (Meng et al., 2016). Previous studies suggested that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner (Lin et al., 2017). As expected, environmental stresses such as osmotic stress, induced cytoplasmic translocation of TEAD and YAP/TAZ (Figure 4).

LIMITATIONS
The preparation of 6% Phos-tag gels in this protocol is only suitable for detecting YAP or TAZ in the Hippo pathway, the molecular weight of which is about 70 kDa. For other proteins of interest, researchers should optimize the acrylamide concentration, Phos-tag concentration, gel electrophoresis and transferring time, to ensure that the phosphorylated form of target proteins can be properly separated. In general, the 6% Phos-tag gel should be selected for those proteins of interest with a molecular weight more than 60 kDa; the 8% Phos-tag gel should be selected for those proteins of interest with a molecular weight less than 60 kDa. However, it is hard to separate proteins with a molecular weight greater than 150 kDa by Phos-tag gels. One advantage of Phos-tag gel analysis is that it only needs antibodies against total proteins to detect the phosphorylation of target proteins, which does not require phosphospecific antibodies. However, if the protein of interest has more than one phosphorylation site, the phos-tag gel cannot provide information about its specific phosphorylation sites. Furthermore, one big advantage of phos-tag is that it provides quantitative information regarding the stoichiometry of phosphorylation. It should also be noted, based on our experience, that not all phosphoproteins are well-resolved by Phos-tag gel.
For in vitro kinase assay for MST1 or MAP4K4, recombinant GST-LATS1 or GST-LATS2 should be used as the substrate. For LATS1 or MAP4K4, the amount of cells from one well of a 6-well plate is generally sufficient to provide enough protein for the kinase analysis described above. However, for MST1, this amount should be scaled up in 100 mm culture dishes. Another limitation is that both the IgG heavy chain and the protein (A+G) could alter the shapes of the 55 kDa protein bands due to the similar molecular weight. The protein (A+G) is easy to fall off from the magnetic beads when Figure 3. In vitro kinase assay of immunoprecipitated LATS1 LATS1 was immunoprecipitated and assayed using GST-YAP as a substrate. GST-YAP phosphorylation was detected by the YAP(S127) phosphospecific antibody. IgG, immunoglobulin G; IP, immunoprecipitation.
boiling, so a pH2.0 buffer can be used to elute the proteins from the magnetic beads. If too much LATS is used in the kinase assay, the substrate GST-YAP might be limiting. Therefore, different amount of immunoprecipitated LATS1 should be tested in the above in vitro kinase assay to ensure that the level of GST-YAP phosphorylation is linearly proportional to the amount of immunoprecipitated LATS1.
The last limitation for YAP or TEAD subcellular localization detection is that it has been optimized for adherent cells. To do immunofluorescence for suspension cells, Thermo Scientific Cytospin 4 Centrifuge needs to be used to attach the suspension cells to slides, which results in forming a uniform monolayer of cells onto an area of a glass slide.

TROUBLESHOOTING Problem 1
No shift band in the YAP Phos-tag gel. (Steps 2 and 12 in Part I)

Potential solution
If only one band of about 70 kDa can be observed in the YAP Phos-tag gel, or the shift of multiple bands is small, it may be due to the ineffective Phos-tag chemical.
One reason is that Phos-tag has been expired or is not stored protected from light. Another reason may be that the 13 SDS-PAGE sample buffer is contaminated with EDTA or EGTA.
A phos-tag molecule requires at least two Mn 2+ to bind with phosphate to exert its effect (Kinoshita et al., 2009). Make sure that the sample does not contain EDTA or EGTA, which binds Mn 2+ thus, interfering the protein mobility shift in Phos-tag gels.

Problem 2
Band dispersion, bending, and tailing. (Steps 2, 3 and 5 in Part I) Figure 4. Immunofluorescence detects TEAD and YAP/TAZ subcellular localization HEK293A cells were treated with or without 200 mM NaCl for 6 h and probed with antibodies that recognize all TEAD isoforms (Pan-TEAD) or YAP/TAZ, and followed by fluorescence-labeled secondary antibodies. DAPI stained for DNA. Scale bars, 10 mm.

Potential solution
Of note, long-time transferring under constant current will cause protein decomposition and dispersion due to high temperature.
You may perform the electrophoresis in a 4 C environment, and pre-cool the transferring buffer sufficiently before use. It is strongly recommended to change the Ice Cooling Unit in the transfer unit during the transferring process for Phos-tag gel to protect the transfer buffer from overheating.
EDTA, inorganic salts, and surfactants are all potential causes of band dispersion or tailing. These impurities in protein samples can be reduced by TCA precipitation or dialysis.
Additionally, neighboring blank wells can also cause bands to bend, which should be filled with the same volume of 13 SDS-PAGE Sample Buffer as the sample wells.

Problem 3
Low resolution of bands in the Phos-tag gel. (Step 4 in BYB)

Potential solution
In general, increase the concentration of Phos-tag could enhance the electrophoretic retardation of phosphoprotein. However, increasing the concentration of Phos-tag will not only result in the slowdown of electrophoretic migration of protein, but also cause band smear and waste this expensive reagent. Usually, 30 mL of 5 mM Phos-tag for two single mini gel (final Con. 15 mM) should work for YAP phosphorylation analysis. The Phos-tag concentration can be increased up to 30 mM.

Problem 4
Low amount of LATS1 protein was recovered. (Steps 13, 14 and 16 in Part II)

Potential solution
Several reasons may reduce the amount of LATS1 protein pulled down.
The LATS1 protein is degraded. Make sure to add Protease/Phosphatase Inhibitor Cocktail into the MLB during step 16.
The number of cells used for LATS1 IP can be scaled up. Although HEK293A cells express a high level of LATS1, LATS1 expression levels in different cell types can vary significantly.
The magnetic beads used in step 14 are not enough. It is noteworthy that the magnetic beads are very easy to aggregate after standing, so they should be thoroughly suspended before each use. Also, never freeze the beads.

Problem 5
Low or no activity of recovered LATS1. (Steps 13, 14, 18 and 20 in Part II)

Potential solution
If the activity of recovered LATS1 is very low (but the amount of LATS1 protein recovered is OK), it is possible that LATS1 is inactive under the particularly context. Cells with high LATS activity should be collected as a positive control, such as high density cultured and/or serum starved cells.
It is also possible that some problems occurred in the LATS1 pull-down process or kinase reaction system, like insufficient amount of cells, primary antibodies or magnetic beads, poor quality ATP in the reaction buffer.
Make sure that phosphatase inhibitors are present in the lysis buffer.