Protease and phosphotase inhibitors cocktail

After different drug treatment, the cells were stimulated with 10 ng/ml EGF for 15 min before harvesting, washed twice with cold PBS and lysed with RIPA buffer that containing protease and phosphotase inhibitors cocktail (Roche, UK). The supernatants were collected after centrifugation at 12000 rpm for 20 min. The protein was applied to polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and then detected by the proper primary and secondary antibodies before visualization with a chemiluminescence kit. The intensity of blot signals was quantitated using ImageQuant TL analysis software (General Electric, UK).

After different treatments, the cells were harvested, washed twice with cold PBS and lysed with RIPA buffer that containing protease and phosphotase inhibitors cocktail (Roche, UK). The supernatants were collected after centrifugation at 12000 rpm for 20 min. The protein was applied to polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and then detected by goat anti-rat cytochrome C antibody (Abcam, USA) and goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (CWBoitech, Beijing, China) before visualization with ChemiDocXRS+ (Biorad, USA). The intensity of blot signals was quantitated using ImageQuant TL analysis software (General Electric, UK).