Cells were lysed with urea buffer (9.5 M urea, 2% CHAPS). Aliquots of cell lysates were heated at 95°C for 5 min and subjected to SDS-polyacrylamide gel electrophoresis. The following primary and secondary antibodies were used anti-Yap antibody (Cell Signaling), anti-actin (Santa Cruz Biotechnology), anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technology), and anti-mouse IgG conjugated with horseradish peroxidase (Cell Signaling Technology)
Anti yap antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-YAP antibody is a research-use-only product that specifically detects the YAP (Yes-associated protein) protein in various biological samples. YAP is a transcriptional co-activator that plays a crucial role in the Hippo signaling pathway, which regulates cell proliferation, differentiation, and apoptosis. The antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to identify and study the expression and localization of YAP in cells and tissues.
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Lab products found in correlation
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Anti rabbit igg conjugated with horseradish peroxidase, by Cell Signaling Technology (2 mentions)
Anti mouse igg conjugated with horseradish peroxidase, by Cell Signaling Technology (2 mentions)
A1 n sim, by Nikon (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Lsm software, by Zeiss (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Anti egfr antibody, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Anti ki67 antibody, by Cell Signaling Technology (1 mentions)
Cy3 conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated goat anti mouse, by Jackson ImmunoResearch (1 mentions)
Ubiquitination kit, by R&D Systems (1 mentions)
Ube2d1, by Sino Biological (1 mentions)
E1 enzyme, by R&D Systems (1 mentions)
E2 enzyme, by R&D Systems (1 mentions)
25 protocols using anti yap antibody
1
Yap Protein Expression Analysis
2
ChIP Assay for Protein-DNA Interaction
3
Immunofluorescence Staining of Cell Proliferation and Mechanosensing
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The cells were seeded on confocal dishes (1 × 105 cells per dish) (Jet Biofil, Guangzhou, China) After treatment, they were fixed with 4% paraformaldehyde-PBS for 15 min, and then permeabilized with 0.1% Triton X-100 in TBS. After blocking with 10% goat serum in PBS for one hour, the cells were incubated with anti-Ki67 antibody (Abcam, CA, USA) or anti-YAP antibody (Cell Signaling, MA, USA) at 4 °C, overnight. After three washes with PBS, the cells were incubated with Alexa Fluor 488 tagged goat anti-rabbit antibody (Cell Signaling, MA, USA, 1:1000 dilution) for 2 h and counterstained with 4′6′-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, MA, USA) for 5 min at room temperature. Immunofluorescence was detected using Zeiss LSM 710 confocal laser scanning microscope with multiphoton System and analysed with Zeiss LSM software (Carl Zeiss, Germany).
4
Western Blot Antibody Incubation Protocol
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Anti-YAP antibody (#14074) and anti-EGFR antibody (#4267) were purchased from Cell Signaling Technology (Beverly MA, USA). The transferred membranes were subsequently incubated overnight (more than 16 hr) at 4 °C with the primary antibody (1:1000) and then the secondary antibody (1:3000) for 1 hr. Chemoluminescence detection was performed by using the Pierce ECL Western Blotting Substrate (Thermo Scientific).
5
Verteporfin and YAP Localization Assay
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Cells were seeded onto Ibidi µ-Slide 8 Well overnight and treated with or without verteporfin or CA3 for 48 h, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton. Samples were incubated with Anti-YAP antibody (Cell signaling Technology, Leiden, The Netherlands). F-actin and nucleus were stained using respectively Alexa-fluor 488 phalloidin and DAPI. Images were acquired using a confocal microscope (NIKON A1 N-SIM) and processed using ImageJ.
6
Immunohistochemical Analysis of Liver Tissue
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Fresh frozen liver tissue was sectioned at 8 μm and stained using previously described methods35 (link). Anti-YAP antibody and anti-Ki67 antibody (Cell Signaling, Beverly, MA) were used for primary incubation. For secondary antibody incubation we utilized Cy3-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). The data was analyzed using open source software from the National Institutes of Health (ImageJ).
7
Yap Protein Expression Analysis
8
Ubiquitination Assay Protocol
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Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were mixing with 20X E1 Enzyme, 10X Mg2+-ATP Solution, 10X Ubiquitin Solution, 1ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer. The reaction was carried out at 37 °C for 1 h and products were analyzed by western-blot assays with anti-YAP antibody (#14074, Cell signaling, 1:1000).
9
Detecting YAP Ubiquitination in Cells
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To directly detect K48 ubiquitinated and total ubiquitinated YAP enriched in cell extracts, HA-K48 ubiquitinated or HA-Ub plasmids, Myc-YAP and Flag-RNF31 or Flag-Vectors were transfected into HEK293T cells. After 24 hours, 10 μM MG132 was added to the cells for treating 8 hours. Then, cells were lysed with Western and IP lysis buffer (P0013J, Beyotime) with a protease inhibitor cocktail (Roche). Total cell lysates were incubated with 30 μl of Protein Agarose (Beyotime, P2012) for preclearing 2 h at 4 °C, and immunoprecipitation was then performed with anti-YAP antibody (14,074, Cell Signaling Technology) or Rabbit IgG (Beyotime, A7016) for 4 h at 4 °C. Western blot was performed with anti-HA antibody to detect YAP total polyubiquitinated or YAP K48 polyubiquitinated.
10
Immunoprecipitation of YAP in HepG2 cells
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To carry out immunoprecipitation (IP), HepG2 cells were treated with 0.1% DMSO (control) or CA for 24 h and lysed in lysis buffer containing 50 mM NaCl, 1 mM ethylene glycol tetraacetic acid, 0.1% SDS, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mg/ml aprotinin, 1 mg/ml leupeptin in 10 mM Tris buffer (pH 7.4), and 1 mM phenylmethanesulfonyl fluoride. The lysates were then sonicated and centrifuged, and the supernatant was incubated with anti-YAP antibody (Cell Signaling Technology) overnight at 4°C. The immunocomplexes were then incubated with PureProteome magnetic beads (Millipore) for 1 h at 4°C, washed, eluted with protein sample buffer (Millipore), and analyzed by Western blotting.
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