Rabbit anti yap1 antibody

HCC specimens and adjacent tissue specimens were cut into 4-µm paraffin-embedded sections, mounted on slides, deparaffinized with xylene and rehydrated with graded ethanol, and blocked with 3% hydrogen peroxide. Heat-induced antigen retrieval was performed using a steamer at 95°C. The primary antibody Rabbit Anti-YAP1 antibody (Abcam, MA, USA) was diluted at a ratio of 1:50. Incubation was performed at 4°C overnight, followed by incubation with a secondary antibody Goat Anti-Rabbit IgG H&L (horseradish peroxidase [HRP]) (Abcam) in 1:50,000 dilution at 37°C for 60 min. Diaminobenzidine was subsequently applied for 10 min. Finally, the sections were counterstained with hematoxylin, dehydrated, covered, and visualized. According to previous studies,12 (link),13 (link) the expression of YAP1 was determined by the density of positive cells and the intensity of staining. The density was scored based on the proportion of positive cells (0: none; 1: ≤10%; 2: 11–25%; 3: 26–50%; 4: >50%), and the intensity was scored based on the average staining intensity of positive cells (0: none; 1: weak; 2: intermediate; 3: strong). The final score was the product of the density score and intensity score, ranging 0–12. Patients were classified into two groups: YAP1 low expression (score: 0–3) and YAP1 high expression (score: 4–12).

Cells were lysed in radioimmunoprecipitation assay lysis buffer (Sigma–Aldrich, MO, USA). After electrophoresis on a 12% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, proteins were transferred onto polyvinylidene difluoride membranes. Subsequently, the membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with the following primary antibodies: Rabbit Anti-YAP1 antibody (1:5000 dilution; Abcam), Rabbit Anti-YAP1 (phosphorylated S127) (pYAP1) antibody (1:50000 dilution; Abcam), Rabbit Anti-Ki67 antibody (1:5,000 dilution; Abcam), Rabbit Anti-TEAD-1 antibody (1:50000 dilution; Abcam), Rabbit Anti-E-cadherin antibody (1:50000 dilution; Abcam), Rabbit Anti-N-cadherin antibody (1:1000 dilution, Abcam), and Rabbit Anti-glyceraldehyde-3-phosphate dehydrogenase (Anti-GAPDH) antibody (1:10000 dilution, Abcam). The HRP-conjugated secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:20000 dilution, Abcam, USA) was added and incubated at room temperature for 1 h. Signals were visualized after chemiluminescence reaction with horseradish peroxidase substrate. The relative protein expressions of YAP1, pYAP1, Ki67, Tead, EMT markers (E-Cadherin and N-Cadherin) in different cells were normalized to the GAPDH concentration, and the quantitative analysis was performed using the Image J software (National Institutes of Health (NIH), USA).