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Phospho yap

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Phospho-YAP is a laboratory reagent used to detect and quantify the phosphorylation of the YAP protein. YAP is a transcriptional co-activator that plays a key role in the Hippo signaling pathway, which regulates cell growth, proliferation, and apoptosis. The Phospho-YAP product allows for the specific detection and measurement of the phosphorylated form of YAP, which is an important indicator of Hippo pathway activity.

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Lab products found in correlation

Phospho lats1, by Cell Signaling Technology (7 mentions) Gapdh, by Santa Cruz Biotechnology (3 mentions) β actin, by Santa Cruz Biotechnology (3 mentions) Total yap, by Cell Signaling Technology (3 mentions) Total lats1, by Cell Signaling Technology (3 mentions) Lats1, by Cell Signaling Technology (2 mentions) Glutamine synthetase, by BD (2 mentions) Phospho β catenin, by Cell Signaling Technology (2 mentions) β catenin, by Cell Signaling Technology (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Phospho akt, by Cell Signaling Technology (2 mentions) Lamin b, by Santa Cruz Biotechnology (2 mentions) Odyssey imaging system, by LI COR (2 mentions) Yap taz, by Cell Signaling Technology (2 mentions) Lats2, by Cell Signaling Technology (2 mentions) Phospho fak, by BD (2 mentions) Phospho src, by Cell Signaling Technology (2 mentions) Phospho erk, by Merck Group (2 mentions) Phospho lats1 2, by Abcam (2 mentions) Phospho mlc2, by Cell Signaling Technology (2 mentions)

27 protocols using phospho yap

1

Immunoblotting Analysis of Hippo Pathway

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Cells or tissues were lysed in RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl at pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF) with protease inhibitors (Roche). The proteins were separated on SDS–polyacrylamide gels and transferred onto PVDF membranes (Millipore). The blots were probed with antibodies against YAP (Cell Signaling, #4912), phospho-YAP (Ser112; Cell Signaling, #4911), phospho-YAP (Ser366; Cell Signaling, #13619), Lats1 (Cell Signaling, #3477), Lats2 (Cell Signaling, #13646), Mst1 (Cell Signaling, #ab51134), NF2 (Sigma, HPA003097), Mst2 (Epitomics, 1943-1), CTGF (Santa Cruz Biotechnology, sc-14939), α-tubulin (Developmental Studies Hybridoma Bank, 12G10), PAN-TEAD (Cell Signaling, #13295), MOB1 (Cell Signaling, #3863), phospho-MOB1 (Cell Signaling, #8699), YAP, and TAZ (Cell Signaling, #8418) and normalized by Actin (Millipore, MAB1501). Signals were detected and quantified by a LI-COR infrared imaging system. The antibodies used for coimmunoprecipitation were 14-3-3 (Santa Cruz Biotechnology, sc-732) and YAP (Novus Biologicals, NB110-58358).
Chen Q., Zhang N., Xie R., Wang W., Cai J., Choi K.S., David K.K., Huang B., Yabuta N., Nojima H., Anders R.A, & Pan D. (2015). Homeostatic control of Hippo signaling activity revealed by an endogenous activating mutation in YAP. Genes & Development, 29(12), 1285-1297.
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2

Cell Signaling Pathway Reagent Procurement

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Most chemicals, including dexamethasone, 3-isobutyl-1-methylxanthine, insulin, saponin, cycloheximide (CHX), verteporfin, crystal violet, anti-FLAG M2 affinity gels, Dulbecco's modified Eagle medium (DMEM), and antibodies against vinculin (V4505), β-actin (A2228), α-tubulin (T5168), and FLAG-tag (F1804) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Opti-MEM, Lipofectamine 2000, Lipofectamine RNAiMAX, bovine serum albumin, goat serum, 4′,6-diamidino-2-phenylindole (DAPI), and Alexa Fluor-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against PIP5Kα (#9693), PIP5Kγ (#3296), phospho-YAP (Ser127; #4911), YAP (#4912), phospho-LATS1 (Ser909; #9157), LATS1(#9153), phospho-TAZ (Ser89; #59971), TAZ (#4883), Merlin (#6995), HA-tag (#3724), Myc-tag (#2278 and #2276), p44/42 mitogen-activated protein kinase (MAPK, #4695), phospho-p44/42 MAPK (Thr202/Tyr204; #8544), p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182; #9211), c-Jun N-terminal kinase (JNK, #9258), phospho-JNK (Thr183/Tyr185; #4668), Akt (#9272), and phospho-Akt (Ser473; #9271) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against lamin B1 (sc-374015), GAPDH (sc-47724), and green fluorescent protein (GFP, sc-9996) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
Le T.P., Nguyen N.T., Le D.D., Anwar M.A, & Lee S.Y. (2023). Lipid kinase PIP5Kα contributes to Hippo pathway activation via interaction with Merlin and by mediating plasma membrane targeting of LATS1. Cell Communication and Signaling : CCS, 21, 149.
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3

Cellular Signaling Pathway Analysis

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The biochemical reagent Nevadensin (#3807) was purchased from Nature Standard (Shanghai, China). MG132 (#HY-13,259) and Sorafenib (#HY-10,201) were purchased from MedChem Express. The following antibodies were Used: YAP (#14074T), phospho-YAP (#13619T), MST1(#3682T), phospho-MST1/2 (49,332 S), LATS1 (#3477T), phospho-LATS1 (#8654S), Merlin (#12888T), phospho-Merlin ( #13281S), Cleaved Caspase-3 (#9611S), Cleaved Caspase-9 (#20750S), Cleaved PARP (#5625S), CDK6 (#13,331), Anti-rabbit IgG (#7074), Anti-mouse IgG (#7076) were purchased from Cell Signaling Technology ; CDK4 (#11026-1-AP), Cylcin B1 (#28603-1-AP) were purchased from proteintech; β-actin ( #TA-09 ) was purchased from ZSGB-Bio; DNA Content Quantitation Assay (Cell Cycle) (#CA1510), Hoechst 33,258 (IH0060), CCK-8 Cell Proliferation and Cytotoxicity Assay Kit was (CA1210) purchased from Solarbio. PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) purchased from TaKaRa. FastStart Universal SYBR Green Master (ROX) (#4,710,436,001) purchased from Roche. Cell-Light EdU Apollo 488 in Vitro Kit (100T) (#C10310-1) purchased from RiboBio.
Shi H., Zou Y., Wang X., Wang G., Gao Y., Yi F., Xu J., Yin Y., Li D, & Li M. (2023). Activating the Hippo pathway by nevadensin overcomes Yap-drived resistance to sorafenib in hepatocellular carcinoma. Discover. Oncology, 14, 83.
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4

Protein Extraction and Analysis from Mouse Liver

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Mice were sacrificed at different time points as described. Liver tissues were removed, snap frozen in liquid nitrogen, and stored at −80 °C until use. For protein extraction, tissues were homogenized in a cold lysis buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.5% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA) containing protease and phosphatase inhibitor cocktails (Roche). After homogenization, the tissue lysates were allowed to solubilize for 1 h at 4 °C with rotation, and then were centrifuged at 19,700g for 30 min at 4 °C. The supernatants were used for immunoblot analyses. Nuclear extract was performed by using nuclear and cytoplasmic extraction kit (Pierce). Western blots were performed following procedure according to different antibodies. 25 to 50 ug of protein was loaded for each lane and transferred to PVDF membrane. Protein expression was analyzed by immunobloting with β-catenin (Cell Signaling), phospho-β-catenin (Cell Signaling), β-actin (Cell Signaling), H3 (Cell Signaling), p21 (BD pharmingen), p53 (kindly provided by Dr. Loning Fu), phospho-Yap (Cell Signaling), Yap (Cell Signaling), Glutamine Synthetase (BD pharmingen). All dilutions are 1:1000, except that H3 antibody dilution is 1:5000 and Glutamine Synthetase antibody dilution is 1:3000. Full gel scans are shown in Supplementary Fig. 11.
Dong B., Lee J.S., Park Y.Y., Yang F., Xu G., Huang W., Finegold M, & Moore D.D. (2015). Activating CAR and β-Catenin Induces Uncontrolled Liver Growth and Tumorigenesis. Nature communications, 6, 5944.
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5

Immunofluorescence Analysis of GBM Markers

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Human GBM and normal brain tissues were obtained from the Cooperative Human Tissue Network (CHTN). Anti-FRMD6 (Sigma), -Lats1/2 (Bethyl Lab), -MST1/2, -YAP, -c-Met, -PDGFRA, -PDGFRB, and -merlin (Santa Cruz), -actin (Sigma), -v5 epitope (Invitrogen), -phospho-Lats1, -phospho-YAP (Cell signaling), -phospho-c-Met (Invitrogen and Santa Cruz), and -phospho-PDGFRA/B (Santa Cruz and R & D Systems) antibodies were used in the experiments. Anti-Ki67 was from the Fisher Scientific. Secondary anti-rabbit Alexa Fluor® 594 and anti-mouse Alexa Fluor® 488 were from Invitrogen.
Xu Y., Wang K, & Yu Q. (2016). FRMD6 inhibits human glioblastoma growth and progression by negatively regulating activity of receptor tyrosine kinases. Oncotarget, 7(43), 70080-70091.
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6

Western Blot Antibody Validation

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Western blot assay was performed according to previously described standard protocol. Primary antibodies were specific for DDAH1 (1:1000; SAB, #37368), phospho-YAP (1:1000; Cell Signaling Technology, #4911), YAP (1:1000; Cell Signaling Technology, #14074), TAZ (1:1000; Cell Signaling Technology, #83669), ALP (1:500; Santa Cruz, #sc-271431), SMAD4 (1:1000; Cell Signaling Technology, #46535), GAPDH (1:5000; Proteintech, #60004-1-Ig), alpha-tubulin (1:5000; Proteintech, #66031-1-Ig), and Histone H3 (1:5000; Proteintech, #17168-1-AP). Secondary anti-mouse/rabbit HRP-conjugated antibodies were subsequently applied.
Xie Z., Hou L., Shen S., Wu Y., Wang J., Jie Z., Zhao X., Li X., Zhang X., Chen J., Xu W., Ning L., Ma Q., Wang S., Wang H., Yuan P., Fang X., Qin A, & Fan S. (2022). Mechanical force promotes dimethylarginine dimethylaminohydrolase 1-mediated hydrolysis of the metabolite asymmetric dimethylarginine to enhance bone formation. Nature Communications, 13, 50.
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7

Protein Extraction and Analysis from Mouse Liver

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Mice were sacrificed at different time points as described. Liver tissues were removed, snap frozen in liquid nitrogen, and stored at −80 °C until use. For protein extraction, tissues were homogenized in a cold lysis buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.5% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA) containing protease and phosphatase inhibitor cocktails (Roche). After homogenization, the tissue lysates were allowed to solubilize for 1 h at 4 °C with rotation, and then were centrifuged at 19,700g for 30 min at 4 °C. The supernatants were used for immunoblot analyses. Nuclear extract was performed by using nuclear and cytoplasmic extraction kit (Pierce). Western blots were performed following procedure according to different antibodies. 25 to 50 ug of protein was loaded for each lane and transferred to PVDF membrane. Protein expression was analyzed by immunobloting with β-catenin (Cell Signaling), phospho-β-catenin (Cell Signaling), β-actin (Cell Signaling), H3 (Cell Signaling), p21 (BD pharmingen), p53 (kindly provided by Dr. Loning Fu), phospho-Yap (Cell Signaling), Yap (Cell Signaling), Glutamine Synthetase (BD pharmingen). All dilutions are 1:1000, except that H3 antibody dilution is 1:5000 and Glutamine Synthetase antibody dilution is 1:3000. Full gel scans are shown in Supplementary Fig. 11.
Dong B., Lee J.S., Park Y.Y., Yang F., Xu G., Huang W., Finegold M, & Moore D.D. (2015). Activating CAR and β-Catenin Induces Uncontrolled Liver Growth and Tumorigenesis. Nature communications, 6, 5944.
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8

Examination of Mst1/2-Akt-YAP Signaling

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Murine recombinant GM-CSF was purchased from ProSpec. CpG DNA (ODN 1826) was purchased from Invivogen. The following chemicals were used: Akt1 inhibitor MK-2206 (AdooQ Bioscience), myc inhibitors 10074-G5 (Cayman) and (+)-JQ1 (Sigma-Aldrich). Antibodies against the following proteins were used in western blot analyses: Mst1 (Cell Signaling, no. 3682), Mst2 (Cell Signaling, no. 3952), phospho-Akt (Cell Signaling, no. 4060), phospho-YAP (Cell Signaling, no. 13008), YAP (Cell Signaling, no. 14074), c-myc (Cell Signaling, no. 13987), Akt (Santa Cruz, sc-1618), GAPDH (Santa Cruz, sc-32233), Lamin B (Santa Cruz, sc-6217), and β-actin (Santa Cruz, sc-47778).
Cho K.M., Kim M.S., Jung H.J., Choi E.J, & Kim T.S. (2019). Mst1-Deficiency Induces Hyperactivation of Monocyte-Derived Dendritic Cells via Akt1/c-myc Pathway. Frontiers in Immunology, 10, 2142.
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9

Analyzing PRPH2 and Hippo Pathway

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The cells were lysed in lysis buffer, and the proteins were separated by SDS-PAGE under reducing conditions. The membrane was blocked in phosphate-buffered saline/Tween-20 containing 5% BSA. Then, antibodies against PRPH2 (Abcam), phospho-YAP (Cell Signalling), YAP (Cell Signalling), phospho-LATS1 (Cell Signalling), LATS1/2 (Cell Signalling), GAPDH (Sigma) and species-specific secondary antibodies were used to incubate the membrane. The secondary antibodies were detected by the Odyssey imaging system (LI-COR). GTPase pull-down assays were performed according to standard procedures as previously described.20 (link)
Dong K., Xue H., Cheng J., Su J., Li D., Zhang J, & Zhang H. (2019). PRPH2 Activates Hippo Signalling and Suppresses the Invasion and Anoikis Inhibition of Laryngeal Cancer. Cancer Management and Research, 11, 10107-10115.
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10

Immunoblotting Workflow for Protein Analysis

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Immunoblotting was performed as described (38 (link)). Briefly, proteins were extracted from cultured cells or homogenized frozen tumor bits using a TissueRuptor (Qiagen, Cat No./ID: 9002755) on ice using RIPA buffer. Protein extraction from 3D/organoid cultures started with digestion of the Matrigel matrix using Cell Recovery Solution (DLW354253, Sigma) for 1 hour on ice. After digestion with Cell Recovery Solution, cells were centrifuged and RIPA buffer was added into the pellets for protein extraction. Antibodies against the following proteins (and clone or catalogue number) were from Cell Signaling: ROR1 (D6T8C), ROR2 (D3B6F), Non-phospho (Active) β-Catenin (Ser45) (D2U8Y), LEF1 (C12A5), Wnt5a/b (C27E8), Dvl2 (30D2), Axl (C89E7), Integrin β2 (D4N5Z), IGFBP3 (D1U9C), YAP/TAZ (D24E4), Phospho-YAP (Ser127) (D9W2I), MST1 (#3682), MST2 (#3952), MOB1 (E1N9D), Phospho-MOB1 (Thr35) (D2F10), LATS1 (C66B5), Phospho-LATS1 (Ser909) (#9157), SAV1 (D6M6X), Phospho-MST1 (Thr183)/MST2 (Thr180) (E7U1D), Merlin (D3S3W), Lamin B2 (D8P3U), Survivin (71G4B7), Phospho-Merlin (Ser518) (D5A4I), Tubulin (#2148). Additional antibodies were against Kif26B (Proteintech, 17422–1-AP), β-Actin (Santa Cruz), GAPDH (Abcam Ab-9485), and Vinculin (Sigma V9131). The primary antibodies were incubated overnight. Gels shown are representative of at least three independent experiments.
Wang K., Ma F., Arai S., Wang Y., Varkaris A., Poluben L., Voznesensky O., Xie F., Zhang X., Yuan X, & Balk S.P. (2023). WNT5a Signaling through ROR2 Activates the Hippo Pathway to Suppress YAP1 Activity and Tumor Growth. Cancer research, 83(7), 1016-1030.
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