Yap1 antibody

Whole cultured cells were homogenized in 0.1% SDS and 1 mM PMSF (phenylmethylsulfonyl fluoride) and centrifuged at 12, 000 g for 10 min. Protein extracts were subjected to SDS-PAGE and analyzed using the following primary antibodies: LEF1 antibody (Abcam, ab137872), YAP1 antibody (Abcam, ab52771), TAZ antibody (Proteintech, 23306-1-AP), E-cadherin antibody(Abcam, ab40772), N-cadherin antibody (Abcam, ab18203) and GADPH antibody (Abcam, ab8245). Then, the membranes were incubated with secondary antibodies (CST,7076,7074) at room temperature for 1 hour. All experiments were performed in triplicate.

Total protein was isolated from cells subjected to different treatments using RIPA lysis buffer (Beyotime Biotechnology) and the concentrations of total protein were determined using a BCA assay kit (BOSTER Biological Technology Co., Ltd.). Protein samples (20 μg) were separated using 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with anti-cleaved-Poly-(ADP-ribose) polymerase (PARP) antibody (1:1000, Abcam), anti-RB transcriptional corepressor 1 (pRB) antibody (1:1000, Abcam), anti-cyclin dependent kinase 6 (CDK6) antibody (1:1000, Abcam), anti-Yes1 associated transcriptional regulator (YAP1) antibody (1:1000, Abcam), anti- connective tissue growth factor (CTGF) antibody (1:1000, Proteintech Group, Inc.), anti-cysteine rich angiogenic inducer 61 (CYR61) antibody (1:1000, Abcam), and anti-GAPDH antibody (1:10,000, Proteintech Group, Inc.) overnight at 4 °C. After washing three times with PBST (PBS with 0.05% Tween20), the membranes were incubated with the secondary antibody (1: 5000, Jackson ImmunoResearch) at 37 °C for 2 h. After five washes with PBST, the protein bands were visualized using a Millipore ECL system (Shanghai Tanon Technology Co. Ltd., Shanghai, China).