Rabbit anti n cadherin antibody

At 48 h after transfection (pcDNA3.1, pcDNA3.1-PCAT1, scrambled siRNA or PCAT1 siRNA), proteins from cells were extracted by using RIPA protein extraction reagent supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The concentration of proteins was measured by a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). In total, 50 μg of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes (Sigma-Aldrich Co., St Louis, MO, USA). The membranes were blocked with 5% skim milk for 1 h at room temperature, and the membranes were incubated with 1:1,000 rabbit anti-N-cadherin antibody, 1:1,000 rabbit anti-vimentin antibody, 1:1,000 rabbit anti-E-cadherin antibody and 1:2,000 rabbit anti-β-actin antibody (Abcam, Cambridge, UK) overnight at 4°C. After washing with TBST buffer, the membranes were incubated with horseradish peroxidase link-coupled secondary antibody at room temperature for 1 h. The ECL kit was used to visualize the bands, and the intensity of the bands was quantified by densitometry (Quantity One software, Bio-Rad).

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).