*4.11. Western Blot Analysis*

Cells were lysed with RIPA lysis buffer containing a phosphatase inhibitor and PMSF. Extracted proteins were subjected to electrophoresis on 10% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked with 5% (w/v) skim milk powder in TBST for 1 h, and incubated with primary antibodies at 4 ◦C overnight, according to the manufacturer's protocol (dilution ratio of all proteins was 1:1000, except for cytochrome c, which was 1:200). After washing, membranes were incubated with peroxidase-conjugated secondary antibodies (dilution ratio was 1:1000), and protein bands were visualized using the hypersensitive enhanced chemiluminescence (ECL) chemiluminescence kit. Densitometric analysis was performed using the Image J software (1.8.0, NIH, US), and the intensity of specific bands was normalized to the GAPDH band.
