*2.12. Western Blotting*

Mitochondrial fraction was isolated using the Mitochondria Isolation Kit for Cultured Cells according to the manufacturer's instructions (Beyotime Institute of Biotechnology, Nanjing, China). For total protein extraction, cells were washed twice with PBS and lysed with RIPA buffer (Beyotime Institute of Biotechnology, Nanjing, China). The homogenates were centrifuged at 12,000× *g* for 15 min at 4 ◦C, the supernatant was then collected. Protein concentrations were evaluated using the BCA method. Then the samples were denatured by boiling with sample buffer for 10 min, loaded to SDS-PAGE gel for separation, and then transferred onto the PVDF membrane. The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with different primary antibodies overnight at 4 ◦C. The membranes were then washed and incubated with secondary antibodies for 1 h at room temperature. The protein bands were visualized using the enhanced chemiluminescence (ECL) kit (Thermo, Waltham, MA, USA) and quantitated using Image J software (National Institutes of Health, USA). The band density of different proteins was normalized to the control.

ND1 (DF4214) antibody was obtained from Affinity Biosciences (Cincinnati, OH, USA). VDAC1 (55259-1-AP) antibody was purchased from Proteintech Group Inc. (Wuhan, China). Drp1 (A2586), AIF (A19536), cy<sup>t</sup> c (A4912), and caspase-3 (A11021) antibodies were purchased from ABclonal Technology (Wuhan, China). Antibody against β-actin (70-ab008-040) was obtained from MultiSciences Biotech Co. (Hangzhou, China).
