Cytochrome c cyt c

After the indicated treatment, the cells were harvested and lysed with ice-cold lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration of the lysates was determined by a Protein Assay Kit (Thermo, Rockford, Illinois, USA), and equal amounts of protein were separated on a 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane. Nonspecific protein binding was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. The membranes were then incubated with specific AMPKα (1:1000), phospho-AMPKα (p-AMPK; Thr172; 1:1000), p62 (1:1000), poly (ADP-ribose) polymerase (PARP), cleaved PARP (1:1000, Cell Signaling Technology, Massachusetts, USA), LC3 (1:1000), phospho-p70 S6 kinase (Thr389; 1:500, Abcam, Massachusetts, USA), cytochrome c (Cyt-c; 1:1000), Bcl-2 (1:1000), Bax (1:1000, Santa Cruz, State of California, USA), and β-actin (1:2000; MultiSciences, Hangzhou, China) primary antibodies overnight at 4 °C. The membranes were washed the next day and incubated with secondary antibodies at room temperature for 1 h. After being washed with phosphate-buffered saline (PBS) three times, the membranes were incubated with secondary antibodies. The immunoblots were detected by the Odyssey Infrared Imaging System, and the data were quantified with ImageJ software.