Western Blot Analysis of Mitochondrial Proteins

Western blot was carried out essentially as described^{30 (link)} with slight modifications. Total protein was extracted using a whole protein extraction kit (Solarbio, Beijing, China), and protein concentrations were measured using a BCA protein assay kit (Solarbio, Beijing, China). Next, total proteins were separated by SDS/PAGE electrophoresis and then transferred to a PVDF membrane (EpiZyme, Shanghai, China) at 25 °C for 1 h. Overnight incubation with the primary antibody at 4 °C was performed, and then goat anti-rabbit IgG (Beyotime, Shanghai, China) was added at 25 °C for 1 h. Finally, the proteins were detected using a developing solution (Thermo Fisher Scientific, USA). The following primary antibodies were used: CST1 (1:1000; ab307416, Abcam, British), MEK1/2 (1:1000; #9122, CST, USA), p-MEK1/2 (Ser217/221) (1:1000; #9121, CST, USA), p44/42 MAPK (Erk1/2) (1:1000; #9102, CST, USA), p-p44/42 MAPK (Erk1/2) (1:1000; #9101, CST, USA), GRIM19 (1:1000; A18071, Abclonal, China), SDHA (1:1000; A16204, Abclonal, China), UQCRC2 (1:1000; A4184, Abclonal, China), COX IV (1:1000; A6564, Abclonal, China), ATP5A1 (1:1000; A11217, Abclonal, China), VDAC1 (1:1000; A19707, Abclonal, China), MMP2 (1:1000; #4022, CST, USA), and β-actin (1:1000; AF5003, Beyotime, Shanghai, China). All experiments were repeated three times.