Lysis buffer radioimmunoprecipitation assay ripa

Cells were lysed with lysis buffer radioimmunoprecipitation assay (RIPA) (Beyotime Institute of Biotechnology, China) and a mixture of protease inhibitors phenylmethanesulfonyl fluoride (PMSF) (Beyotime Institute of Biotechnology, China). The protein concentration was estimated by bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After being transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA), membranes were blocked with 5 % non-fat milk and then incubated with primary antibodies at 4 °C overnight. Secondary antibodies were incubated for 2 h at room temperature. Protein bands were visualized using the enhanced chemiluminescence detection kit (Thermo scientific, USA). The primary antibodies were: CNTN-1 mAb (1:1000, Abcam, UK), E-cadherin rabbit mAb (1:1000, Cell Signaling Technology, USA), Slug rabbit mAb (1:1000, Cell Signaling Technology, USA), Snail rabbit pAb (1:1000, Abcam, UK), N-cadherin rabbit mAb (1:1000, Cell Signaling Technology, USA) and GAPDH mAb-HRP (1:5000, Bioworld Technology, USA).