Anti phospho yap ser127

Manufactured by Cell Signaling Technology

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Anti-phospho-YAP (Ser127) is a laboratory reagent used to detect the phosphorylation of the transcriptional co-activator YAP at serine 127. This antibody can be used in various immunoassay techniques, such as Western blotting, to study the regulation of YAP signaling.

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Anti-YAP (150 citations) Phospho-YAP (27 citations) Phospho-YAP (Ser127) (21 citations) Anti-phospho-YAP (17 citations) Rabbit anti‐phospho Yap Ser127 (6 citations) Anti-Phospho-YAP (Ser127) (13008), (2 citations) Rabbit polyclonal anti‐phospho‐YAP (Ser127), (2 citations) Anti-phospho YAP (Ser127) (#4911), (2 citations)

19 protocols using anti phospho yap ser127

Profiling YAP Signaling Pathway

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Whole cell lysates were prepared with radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling) containing PhosSTOP and Complete Protease Inhibitor Cocktail (both Roche). Cell lysates were incubated on ice and then subjected to SDS‐PAGE. Blots were probed with anti‐YAP (Cell Signaling #4912, 1:2000), anti‐phospho‐YAP (Ser 127) (Cell Signaling #4911; 1:2000), anti‐GAPDH (Sigma #SAB1405848; 1:5000), anti‐ubiquitin (Sigma #U5379; 1:100), anti‐Plk2 (Sigma #SAB4500156; 1:5000) and anti‐FLAG (Sigma #F3165; 1:2000). Quantification was performed with Image J software.

Mochizuki M., Lorenz V., Ivanek R., Della Verde G., Gaudiello E., Marsano A., Pfister O, & Kuster G.M. (2017). Polo‐Like Kinase 2 is Dynamically Regulated to Coordinate Proliferation and Early Lineage Specification Downstream of Yes‐Associated Protein 1 in Cardiac Progenitor Cells. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(10), e005920.

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Western Blot Analysis of Yap-Hippo Pathway

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Cellular or tissue samples were lysed with RIPA lysis buffer freshly adding cocktail protease inhibitor (Thermo Scientific). Equal amounts of cellular proteins were subjected to electrophoresis in SDS-PAGE, and transferred to nitrocellulose membranes (Millipore). The membranes were blocked and then incubated at 4 °C overnight with indicated first antibodies, followed by incubation with the appropriate HRP-conjugated secondary antibodies (Thermo Scientific). The protein bands were detected and analyzed with an ECL. The following antibodies were used: anti-Aurora A (Upstate, #07-648), anti-phospho-Aurora A (Thr288) (Cell Signaling Technology, #3079), anti-YAP (Cell Signaling Technology, #4912), anti-phospho-YAP(ser127) (Cell Signaling Technology, #4911), anti-phospho-YAP(Ser397) (Cell Signaling Technology, #13619), anti-Lats1 (Cell Signaling Technology, #3477 P), anti-phospho-Lats1 (Thr1079) (Cell Signaling Technology, #9654P), anti-Lats2 (Rui Ying biological, China, #RLP1047), anti-SAV1 (Cell Signaling Technology, #3507P), anti-MST1 (Cell Signaling Technology, #3682P), anti-GAPDH (Kangcheng, China,#KC-5G4), anti-Lamin B1 (Epitomics, #6581-1), anti-CTGF (Life Science Products & Services, #AB60212a), anti-Myc Tag (MERCK, 05-724).

Wang P., Gong Y., Guo T., Li M., Fang L., Yin S., Kamran M., Liu Y., Xu J., Xu L., Peng F., Xue X., Yang M., Hung M.C., Lam E.W., Gu C., Wang C., Zhan Q, & Liu Q. (2019). Activation of Aurora A kinase increases YAP stability via blockage of autophagy. Cell Death & Disease, 10(6), 432.

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Western Blot Analysis of Cell Signaling Proteins

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RIPA lysis buffer (Boston Bio-Products, MA) was used for protein extraction from cell lines. The Halt^™ Protease Inhibitor Cocktail, EDTA-free (100X) (Life Technology; MA) was added before harvesting protein lysates. 40 μg protein lysates were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes (EMD Millipore; MA) and blocked with non-fat milk for 1 hr. Primary antibodies were prepared in 5% BSA or non-fat milk and incubated with PVDF membrane at 4 °C overnight. The HRP-conjugated goat anti-mouse (Bio-Rad # 1706516) or goat anti-rabbit (Bio-Rad # 1706515) secondary antibodies were incubated with membrane for 2 hours. ECL Plus Western Blotting Detection Reagents (GE Healthcare; PA) were used for protein detection. Anti-NTRK1 (# 2505), anti-YAP (#14074), anti-phospho-YAP (Ser127) (# 13008), anti-LATS1 (# 9153), anti-phospho-LATS 1 (Ser909) (# 9157), anti-MST1 (# 14946) and anti-phospho-MST1/2 (T183/T180) (#3681) antibodies from Cell Signalling Technologies (Beverly, MA); anti-GAPDH (#Y1041) antibody from Ubiquitin-Proteasome Biotechnologies (Aurora; CO); anti-Flag M2 antibody was purchased from Sigma-Aldrich (#F1804–1MG);

Yang X., Shen H., Buckley B., Chen Y., Yang N., Mussell A.L., Chernov M., Kobzik L., Frangou C., Han S, & Zhang J. (2018). NTRK1 is a positive regulator of YAP oncogenic function. Oncogene, 38(15), 2778-2787.

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Antibody Validation for Immunoblotting

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The following antibodies were used in this study (with dilution factor for immunoblotting): anti-PKN2 (#2612, 1:1,000), anti-YAP (#14074, 1:1,000), anti-YAP/TAZ (#8418, 1:1,000), anti-phospho-YAP (Ser127) (#4911, 1:1,000), anti-phospho-EGFR (Tyr1068) (#3777, 1:1,000), anti-EGFR (#4267, 1:20,000), anti-phospho-AKT (Ser473) (#4058, 1:1,000), anti-AKT (#9272, 1:5,000), anti-phospho-ERK1/2 (Thr202/Tyr204) (#9101, 1:1,000), anti-ERK1/2 (#9102, 1:5,000), anti-ARIH2/TRIAD1 (#13689, 1:1,000), anti-BTAF1 (#2637, 1:1,000), anti-GNAQ (#14373, 1:1,000), anti-HSP90 (#4877, 1:5,000), anti-ALDOA (#8060, 1:5,000), anti-METAP2 (#12547, 1:1,000), anti-GAPDH (#2118, 1:5,000), anti-RHOA (#2117, 1:1,000), anti-Cofilin (#5175, 1:1,000), anti-phospho-Cofilin (Ser3) (#3313, 1:1,000), from Cell Signaling Technology; anti-α-Tubulin (T6074, 1:20,000) and anti-β-Actin (A1978, 1:20,000) from Sigma; anti-GNB2 (ab81272, 1:1,000), anti-RIC8A (ab97808, 1:1,000), anti-USP22 (ab195289, 1:1,000), anti-CUL5 (ab184177, 1:1,000), anti-RNF7 (ab181986, 1:1,000), anti-PDCD10 (ab180706, 1:1,000), from Abcam; anti-KCTD5 (#15553–1-AP, 1:1,000), anti-PSAT1 (#10501–1-AP, 1:5,000), from Proteintech; Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugate (#AP307P, 1:5,000), Goat Anti-Mouse IgG Antibody, (H+L) HRP conjugate (#AP308P, 1:5,000), from Millipore Sigma.

Zeng H., Castillo-Cabrera J., Manser M., Lu B., Yang Z., Strande V., Begue D., Zamponi R., Qiu S., Sigoillot F., Wang Q., Lindeman A., Reece-Hoyes J.S., Russ C., Bonenfant D., Jiang X., Wang Y, & Cong F. (2019). Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC. eLife, 8, e50223.

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Palmitate and LPC Immunohistochemistry

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Palmitate (PA) (P0500) was obtained from Sigma-Aldrich (St. Louis, MO). LPC (Sigma, St. Louis, MO) was dissolved as previously described (8 (link)). Primary antisera employed for the studies include anti-alpha smooth muscle actin (αSMA) (ab124964) and anti-fibronectin (ab2413) antibodies from Abcam (Cambridge, MA), anti-GAPDH (MAB374) from Millipore Sigma, anti-β-actin (sc-47778) from Santa Cruz Biotechnologies (Santa Cruz, CA) and anti-Lyve1 (AF2125) from R&D systems (Minneapolis, MN), anti-F4/80 (70076) and anti-phospho Yap (Ser127) (4911) from Cell Signaling Technology (Danvers, MA), anti-Yap1 (sc-101199) from Santa Cruz Biotechnologies (Santa Cruz, CA), anti-type I collagen antibody (1310-01) from Southern Biotech. Recombinant human VCAM1 (rhVCAM1) (862-VC) was obtained from R&D systems.

Guo Q., Furuta K., Islam S., Caporarello N., Kostallari E., Dielis K., Tschumperlin D.J., Hirsova P, & Ibrahim S.H. (2022). Liver sinusoidal endothelial cell expressed vascular cell adhesion molecule 1 promotes liver fibrosis. Frontiers in Immunology, 13, 983255.

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Investigating YAP Signaling in H. pylori Infection

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Antibodies and their sources were as follows: YAP inhibitor verteporfin from Sigma-Aldrich (St. Louis, MO, USA) for western blot assay, Anti-GAPDH (#2118), Anti-YAP (#4912), Anti-Phospho-YAP Ser127 (#4911), and Anti-Slug (#9585), from Cell Signaling Technology (Beverly, MA, USA); Anti-TAZ (HPA007415) from Sigma (St. Louis, MO, USA) (Anti-E-cadherin (#610405), Anti-N-cadherin (#610921) from BD Biosciences (San Jose, CA USA); Anti-CagA (sc-28,368) and Anti-phospho-tyrosine (sc-7020) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-H. pylori urease B (ab127916) from Abcam (Cambridge, MA, USA). For immunohistochemistry assay, Anti-YAP (#4912) from Cell Signaling Technology, Anti-TAZ (HPA007415) from Sigma, Anti-E-cadherin (#610405) from BD Biosciences For immunofluorescence assay, Anti-YAP (#4912) from Cell Signaling Technology, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies from Invitrogen (Thermo Fisher Scientific, Suwanee, GA, USA). The recombinant plasmid of YAP CDNA, CagA were constructed and purchased from GeneChem, Shanghai, China. YAP siRNA was purchase from Santa Cruz Biotechnology.

Li N., Feng Y., Hu Y., He C., Xie C., Ouyang Y., Artim S.C., Huang D., Zhu Y., Luo Z., Ge Z, & Lu N. (2018). Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway. Journal of Experimental & Clinical Cancer Research : CR, 37, 280.

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Western Blot Analysis of Cell Signaling

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Same numbers of cells were used to extract proteins in 1× passive lysis buffer (Promega; E194A), and protein supernatant was collected after centrifugation at 16,200 g at room temperature. Proteins were denatured in 1× sample buffer (10% glycerol, 0.002% bromophenol blue, 0.075 M Tris, 2% SDS and 5% beta-mercaptoethanol) by boiling at 99°C for 10 min and centrifuged at 16,200 g for 10 min. Western blotting was performed as described (Kumar et al, 2013 (link)). Following antibodies were used for immunoblot analyses: anti-phospho p53 (s15) (Cell Signaling; #9284), anti-phospho p38 MAPK (Thr 180/Tyr 182) (Cell Signaling; 4631), anti-phospho-YAP (Ser127) (Cell Signaling; #4911), anti-p21 (H-164) sc-756 (Santa Cruz Biotech) and anti-GAPDH (Bethyl; A300-641A).

Meena J.K., Cerutti A., Beichler C., Morita Y., Bruhn C., Kumar M., Kraus J.M., Speicher M.R., Wang Z.Q., Kestler H.A., d’Adda di Fagagna F., Günes C, & Rudolph K.L. (2015). Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion. The EMBO Journal, 34(10), 1371-1384.

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Rat Brain Protein Isolation and Analysis

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Rat brain proteins were isolated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to a BioTrace™ NT nitrocellulose transfer membrane (#66485, Pall, CO, New York, USA). The membrane was blocked at room temperature for 2 h with 5% skim milk or bovine serum albumin and then incubated overnight at 4°C with the following primary antibodies: anti‐CXCR7 (1:100, rabbit, NBP2‐24779, Novus Biologicals, Littleton, CO, USA), anti‐TCF7L2 (1:500, rabbit, A19548, ABclonal, Wuhan, China), anti‐phospho‐TAZ resistant (Ser89) (1:1000, rabbit, 59971 S, Cell Signaling Technology, Danvers, MA, USA), anti‐phospho‐YAP (Ser127) (1:1000, rabbit, 13,008 T, phospho‐Yap (Ser127), Cell Signaling Technology, Danvers, MA, USA), anti‐β‐actin (1:500, rabbit, AF5006, Beyotime, Shanghai, China), and anti‐GAPDH (1:2000, AF1186, Beyotime, Shanghai, China). After washing with TBST three times, the membrane was incubated with secondary antibodies for 12 h and detected with a chemiluminescence HRP substrate solution (#WBKLS0050, Merck‐Millipore, Burlington, MA, USA). Blotting was analyzed using a ChemiDoc XRS system (Bio‐Rad, Hercules, California, USA). The ImageJ software was used to quantify the gray values of the protein fragments. For the loading control, all values were normalized to those of β‐actin or GAPDH, which was used as an internal control.

Wang Q., Xu S., Wang B., Qin Y., Ji Y., Yang Q., Xu Y, & Zhou Z. (2022). Chemokine receptor 7 mediates miRNA‐182 to regulate cerebral ischemia/reperfusion injury in rats. CNS Neuroscience & Therapeutics, 29(2), 712-726.

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Western Blot Analysis of Protein Phosphorylation

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Proteins were extracted using Cell Extraction Buffer (Invitrogen) supplemented with phosphatase inhibitors (Roche), protease inhibitors (Roche), and PMSF (Sigma). Protein concentrations were measured by BCA protein assay. 25μg of protein was loaded to each lane for migration by electrophoresis in 4–20% polyacrylamide gel (Bio-Rad). Proteins were transferred to nitrocellulose membrane (Bio-Rad) and blocked before staining. Anti-phospho YAP ser 127 (Cell Signaling), anti-YAP (Cell Signaling), anti-agrin (EMD Millipore), and anti-GAPDH (Cell Signaling) antibodies were used. Membranes were stripped in mild stripping buffer before reprobing with pan antibody or loading control. Horseradish peroxidase conjugated secondary antibody (Cell Signaling) was used for signal detection. The intensity of each band was measured using ImageJ to quantify protein amount.
Explants were washed in PBS and flash froze or lysed immediately. Explants were homogenized in Cell Extraction Buffer using a bead blender, and followed by western blot.

Wang X., Senapati S., Akinbote A., Gnanasambandam B., Park P.S, & Senyo S.E. (2020). Microenvironment stiffness requires decellularized cardiac extracellular matrix to promote heart regeneration in the neonatal mouse heart. Acta biomaterialia, 113, 380-392.

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Western Blot Analysis of EMT Markers

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The proteins isolated from cells were separated using 10% SDS-PAGE and then transmitted to nitrocellulose (NC) membranes (Sigma, San Francisco, CA, USA). After that, the membranes were incubated with primary antibodies overnight, followed by incubation of horseradish peroxidase (HRP)-conjugated secondary antibodies (no. ab6728, 1:5,000; Abcam, UK) for another 1 h. The primary antibodies used in this study are as follows: anti-E-cadherin (no. 14472, 1:1,000), anti-β-catenin (no. 8480, 1:1,000), anti-N-cadherin (no. 13116, 1:1,000), anti-hnRNP-K (no. 9081, 1:1,000), anti-YAP (no. 14074, 1:1,000), anti-phospho-YAP (Ser¹²⁷) (no. 13008, 1:1,000), anti-TAZ (no. 70148, 1:1,000), anti-phospho-TAZ (Ser⁸⁹) (no. 59971, 1:1,000) (all of the above were purchased from Cell Signaling Technology, Boston, MA, USA); anti-Vimentin (sc-66002, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-ZEB1 (A301-921A, 1:2,000; Bethyl Laboratories, USA); and anti-GAPDH (G5262, 1:1,000; Sigma-Aldrich). GAPDH served as a normalized control, and proteins were captured using SuperSignal chemiluminescence substrate (Pierce, Thermo Scientific, Waltham, MA, USA) and analyzed by ImageJ (National Institutes of Health, USA).

Ji L., Li X., Zhou Z., Zheng Z., Jin L, & Jiang F. (2019). LINC01413/hnRNP-K/ZEB1 Axis Accelerates Cell Proliferation and EMT in Colorectal Cancer via Inducing YAP1/TAZ1 Translocation. Molecular Therapy. Nucleic Acids, 19, 546-561.

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