Goat rabbit

The proteins of mitochondria are important components of the apoptotic pathway. C26 cells (1×10⁵ cells per well in 6-well plates) were transfected with the mBim/DMP-039 complex. After 48 hours, cells were collected, and protein samples were extracted with RIPA/IP (Beyotime). In order to ensure that the loading volume for each protein sample was consistent, it was necessary to determine the concentration of total proteins with Coomassie brilliant blue G-250 (Beyotime). Next, the proteins were separated by polyacrylamide gel electrophoresis (Beyotime) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was blocked with 5% non-fat milk (Servicebio Technology, Wuhan). Then we probed the target protein with the primary antibody overnight at 4°C as follows: Bim (1:1000, Abcam), Bcl-2 (1:2000, Abcam), cytochrome c (1:5000, ab133504), caspase-9/cleaved caspase-9 (1:2000, Abcam), and β-actin (1:1000, Abcam). The PVDF membrane was washed and incubated with goat-rabbit (1:10,000, Cell Signaling Technology) secondary antibody. The target proteins were detected using a chemiluminescence system (ChemiScope Touch, CLINX, Shanghai).