Nr5a1

The cell total protein was isolated using RIPA (Applygen Technologies Inc., Beijing, China). Protease inhibitor (CWBIO, Shanghai, China) was added into the RIPA at a ratio of 1:100. After adding RIPA to the cell culture plate, we collected the cells and centrifuged (12,000 rpm) the material at 4 ℃ for 10 min (27) . Protein concentrations were measured on a Thermo Scienti c Pierce BCA protein assay kit (Thermo Fisher, USA) with 1/4 volume of 5 × loading buffer added to the supernatant. A total of 20 µL of protein was blotted using 10% SDS-polyacrylamide gel, then transferred to a polyethylene di uoride (PVDF) membrane (CST, Boston, MA, USA). After blocking with 5% defatted milk for 2 h, the membranes were incubated overnight at 4 ℃ with antibodies (1:1000) against StAR, CYP19A1, CYP11A1, Mfn2, NR5A1 (Abcam, Cambridge, UK) and against Cyclin B, Cyclin D, Cyclin E, CDK4 (Santa Cruz, TX, USA). The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG secondary antibodies (BOSTER, China) were diluted 1:3000 according to the instructions and incubated for 1 h. Detection was performed using chemiluminescence Western blotting substrate (Santa Cruz, CA, USA) in Image Lab analysis software (Image Lab™, Bio-Rad, Berkeley, CA, USA).