A19707

The cells or tissues were treated with RIPA lysate, properly mixed, centrifuged, and the supernatant was added to electrophoresis loading buffer. The separated proteins in the gel were transferred to PVDF membranes, coated with primary antibody overnight, incubated with secondary antibody the next day, and then exposed and developed using a gel imaging system. Primary antibodies used were anti-VDAC1 (A19707; Abclonal), anti-β-Catenin (A0316; Abclonal), phosphorylated β-catenin (AP1076; Abclonal), anti-Cyclin D1 (A0310; Abclonal), anti-GAPDH (A19056; Abclonal).

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.