The 293T cells were transfected with the indicated plasmids using Lipofectamine 2000 Reagent (Thermo Fisher Scientific). After 48 h, cells were collected, and immunoprecipitation and Western blot assay were performed as previously described (50 (link)). For the endogenous interaction between YAP and p65, ATL cell lysate was incubated with anti-YAP antibody (Cell Signaling Technology) or anti-mouse immunoglobulin G (IgG) (Santa Cruz Biotechnology), and the following procedures were the same as described above in 293T cells. Other antibodies used were as follows: anti-FLAG M2, anti-HA, anti–c-Myc (Sigma-Aldrich), anti-p65, anti–phospho-YAP (Ser127), anti–phospho-YAP (Ser381), anti-GAPDH (Cell Signaling Technology), and anti-Tax was used as previously described (51 (link)).
Anti phospho yap
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-YAP is a lab equipment product that detects the phosphorylated form 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 anti-phospho-YAP product can be used to monitor the activation state of YAP in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti yap, by Cell Signaling Technology (10 mentions)
Anti phospho ampkα, by Cell Signaling Technology (5 mentions)
Anti gapdh, by Cell Signaling Technology (5 mentions)
Anti phospho lats1, by Cell Signaling Technology (4 mentions)
Anti lats1, by Cell Signaling Technology (3 mentions)
Anti phospho acetyl coa carboxylase, by Cell Signaling Technology (3 mentions)
Anti taz, by Cell Signaling Technology (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti phospho liver kinase b1 lkb1, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti rabbit igg, by Enzo Life Sciences (2 mentions)
Hrp conjugated anti mouse igg, by Enzo Life Sciences (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Anti phospho lkb1, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Sample buffer, by Bio-Rad (2 mentions)
Anti yap, by Novus Biologicals (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
17 protocols using anti phospho yap
1
Immunoprecipitation and Western Blot of YAP and p65
2
Investigating MUC1-C Protein Interactions in Cell Lysates
3
Molecular Mechanisms of SC Herb in Metabolic Regulation
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Anti-SREBP-1c, anti-phospho-AMPKα, anti-phospho-acetyl-CoA carboxylase (ACC), anti-phospho- liver kinase B1 (LKB1), anti-phospho-YAP, anti-YAP, anti-phospho-large tumor suppressor kinase 1 (LATS1), anti-heme oxygenase 1 (HO-1), anti-Lamin A/C, and anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Nrf2 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Compound C (Com C), verteporfin (VP), T0901317 (T090), Harris hematoxylin and eosin, and Oil Red O solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol (PEG) 400 was obtained from Yakury Pure Chemical Co., Ltd. (Kyoto, Japan). SC is a medicinal-standard herb manufactured at a GMP facility (Nonglim Saengyak, Seoul, Republic of Korea) certified by the Korean FDA and was prepared and standardized as previously described [13 (link)].
4
Antibody Catalog for Cell Signaling
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Rabbit polyclonal anti-Angiomotin was previously described (Yi et al., 2011 (link)) (IB: 1:1500). The following antibodies are available commercially: anti-phospho-Angiomotin (ABS1045 from EMD Millipore; 1:2000). Anti-HA tag (sc805, 1:1000); monoclonal anti-Merlin (E-2, 1:500); polyclonal anti-Merlin (C-18, 1:500); polyclonal anti-YAP (H-125, 1:1000); polyclonal anti-Lamin A/C (sc-6215 (N-18), 1:500); polyclonal anti-EGFR (sc03, 1:1000) were from Santa Cruz Biotechnologies. Anti-Flag tag (F1804, 1:1000); anti-GAPDH (68795, 1:10000); anti-tubulin (T5168, 1:1000); anti-actin (A4700; 1:10000) were from Sigma. Anti-V5 tag (ab27671, 1:2000) from Abcam. Anti-6x His tag (MA1-21315, 1:2000) from Thermo Scientific. Anti-phospho-YAP (4911, 1:1000); anti-pan-Tead (13295; 1:1000) from Cell Signaling Technologies. Anti-Na+/K+ATPase (a-5, 1:2500) from Developmental Studies Hybridoma Bank (University of Iowa). For immunofluorescence the following antibodies were used at the stated concentrations: rabbit monoclonal anti-YAP D8H1X (14074, 1:50), Cell Signaling Technologies; mouse monoclonal anti-Merlin (E-2; 1:50), Santa Cruz Biotechnologies; Rabbit polyclonal anti-Angiomotin (1: 200).
5
Western Blot Analysis of Proteins in HCC
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RIPA Buffer (WB009A; Hat Biotechnology, Xi’an, China) was used to extract proteins in HCC cells or tissues. A BCA kit (WB003; Hat Biotechnology) was used to measure protein concentrations. All proteins were then electrophoresed in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Bio-Rad). The membranes were blocked with 10% non-fat milk, then incubated with specific primary antibodies at 4°C overnight. The antibodies used in the study were anti-LATS2 (1:500; bs-4081R; Beijing Bioss Biotechnology), anti-YAP (1:1000; #14,074; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-YAP (ser127; 1:1000; #13,008; Cell Signaling Technology), anti-thioredoxin-interacting protein (1:1000; #14,715; Cell Signaling Technology), and anti-glyceraldehyde 3-phosphate dehydrogenase (1:1000, #5174; Cell Signaling Technology). The membranes were then incubated with secondary antibody (anti-rabbit #7074 or anti-mouse #7076; Cell Signaling Technology) for 2 h at room temperature. Lastly, enhanced chemiluminescence reagent (PierceTM ECL, Thermo ScientificTM, Waltham, MA, USA) was applied to detect the proteins.
6
Protein Extraction and Western Blot Analysis
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Whole livers and cell lines were lysed with radioimmunoprecipitation buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1 mM EDTA) containing protease and phosphatase inhibitors (1 mM NaF, 1 mM Na3OV4, PMSF, 2 μg ml−1 leupeptin and pepstatin; all purchased from Sigma) for 30 min at 4 °C. The protein concentration in each lysate was quantified with a Pierce BCA protein assay kit (Thermo Scientific). Blots were incubated with anti-LATS1 (Bethyl Laboratories, 1:2,000), anti-LATS2 (Cell Signaling, 1:1,000), anti-phospho-YAP (Cell Signaling (S127), 1:1,000), anti-YAP (Cell Signaling, 1:1,000), anti-TAZ (Cell Signaling, 1:1,000), anti-CTGF (Santa Cruz, 1:500), anti-β-ACTIN (Sigma, 1:5,000), anti-phospho-CHK2 (Cell Signaling, 1:500), anti-phospho-CDC2 (Cell Signaling, 1:500) and p21 WAF1/CIP1 (BD Bioscience, 1:500). We included uncropped blots in Supplementary Fig. 9 .
7
Western Blot Analysis of YAP and Phospho-YAP
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Western blotting was performed as described previously [18 (link)] using the following antibodies: anti-YAP (Cell Signaling Technology), anti-phospho-YAP (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology). To detect the signals, appropriate horseradish peroxidase-conjugated secondary antibodies were used and visualized with PierceTM ECL Western blotting substrate (Thermo Fisher).
8
Western Blot Analysis of Smad2, YAP, and p-YAP
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Cells were lysed in RIPA buffer (1% NP40, 0,5% deoxycholate, 0,1% sodium dodecyl sulfate in Tris-buffered saline (TBS)) (Sigma-Aldrich) supplemented with protease (Complete, Sigma-Aldrich) and phosphatase (PhosSTOP, Sigma-Aldrich) inhibitors. Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk powder in TBS. Primary antibodies used for western blotting were: anti-Smad2 (1/500, Abcam, Cambridge, UK, ab228765), anti-YAP (1/1000, Cell Signaling Technology, Danvers, MA, USA, D8H1X), anti-phospho-YAP (1/1000, Cell Signaling Technology, D9W2I) and anti-GAPDH (1/1000, Cell Signaling Technology, D16H11). Secondary antibody was anti-IgG rabbit (1/5000, Dako, Santa Clara, CA, USA, P0448). Detection was carried out using peroxidase-conjugated antibodies and SuperSignalTM West Femto (Thermo Fisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with ImageJ software (NIH, Bethesda, MD, USA).
9
Spatholobi Caulis Bioactivities Analysis
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Anti-phospho-acetyl-CoA carboxylase (ACC), anti-phospho-AMPKα, anti-phospho-LKB1, anti-Phospho-LATS1, anti-phospho-YAP, anti-YAP, anti-Poly (ADP-ribose) polymerases (PARP), anti-caspase-3, anti-B-cell lymphoma-extra-large (Bcl-xL), and anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were bought from Enzo Life Sciences (Farmingdale, NY, USA). Polyethylene glycol (PEG) 400 is manufactured by Yakury Pure Chemical Co., Ltd. (Kyoto, Japan). SC was produced by water extraction of the medicinal herb, Spatholobi Caulis (Daewon pharmacy, Daewon, Korea) as previously described [17 (link),19 (link)]; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), rhodamine 123 (Rh123), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), arachidonic acid (AA), ferric nitrilotriacetic acid (iron), Harris hematoxylin and eosin (H&E), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
10
Emodin-Induced Metabolic Regulation
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Emodin, dimethylsulphoxide (DMSO), ferric nitrate, nitrilotriacetic acid, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT), rhodamine 123 (Rh123), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). Anti-PARP, anti-procaspase-3, anti-Bcl-xL, anti-phospho-acetyl-CoA carboxylase (ACC), anti-AMPKα, anti-phospho-AMPKα, anti-LKB1, anti-phospho-LKB1, anti-Yes-associated protein (YAP), and anti-phospho-YAP antibodies were obtained from Cell Signalling Technology (Beverly, MA, USA).
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