Cytochrome c

Cells lines were lysed using 1× RIPA Lysis (Solarbio, China) containing PMSF (1:100, Solarbio, China). Protein samples were quantified using the BCA protein assay kit (Solarbio, China) as manufacturer’s instructions. We loaded 20 μg protein per well into 10–12% gels and then transferred it to PVDF membranes using the Trans-Blot Turbo transfer system (Bio-Rad) according to the manufacturer’s instructions. Membranes were blocked for 1 h in 5% BSA and stained with primary antibody overnight at 4 °C. The following primary antibodies were tested: Bcl-xL (1:1000, Abcam), Bcl-xS (1:1000, GeneTex), SQSTM1 (1:1000, Cell Signaling), LC3 (1:1000, Abcam), Beclin-1 (1:1000, Abcam), Cleaved-Caspase3 (1:1000, Cell Signaling), Caspase3 (1:1000, Cell Signaling), Cytochrome c (1:1000, Immunoway). BCL2 (1:1000, Proteintech), MCL1 (1:1000, Proteintech). After washing the blots three times for 10 min each with TBST, the secondary antibody anti-rabbit horseradish peroxidase (1:10000, Immunoway) or anti-mouse horseradish peroxidase (1:10000, Immunoway) was added for 1 h at room temperature. Following another round of 3 × 10 min washes, the membranes were imaged on the Alpha Innotech followed by quantification using Image J.