Caspase 8

Total protein was assessed via SDS-polyacrylamide gel electrophoresis under reducing conditions on 12% gels and then was transferred to nitrocellulose membranes via tank transfer at 80 mV in Tris-glycine buffer containing 20% methanol for 12 h at 4°C. Nitrocellulose membranes were blocked for 1 h with 5% bovine serum albumin (BSA) at 37°C and were incubated for 12 h with the following antibodies at 4°C: PI3K (1:500, Immuno Way, China), AKT (1:500, Immuno Way, China), mTOR (1:500, Immuno Way, China), LC3 (1:1000, Immuno Way, China), Beclin1 (1:500, ImmunoWay, China), BCL2 (1:500, ImmunoWay, China), BAX (1:500, ImmunoWay, China), Caspase3 (1:500, ImmunoWay, China), Caspase8 (1:500, ImmunoWay, China). PBST was used to wash the nitrocellulose membranes 4 times, for 15 minutes each time. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody against rabbit IgG (1:5000, Santa Cruz, CA, USA) using an ECL kit (Kangweishiji Biotechnology, Beijing, China). Then nitrocellulose membranes were washed 4 times using PBST, each time for 15 minutes. The GAPDH content was analyzed as the loading control with rabbit polyclonal antibody (Sigma, USA).