Gapdh bs 2188r

U0126 (inhibitor of MEK1/2; MCE, NJ, USA) was used at a final concentration of 10 μM. GAPDH (bs‐2188r; Bioss, Beijing, China), vimentin (10366‐1‐AP; Proteintech, Wuhan, China), β‐catenin (51067‐2‐AP; Proteintech), E‐cadherin (20874‐1‐AP; Proteintech), PD‐L1 (66248‐1‐Ig; Proteintech), PD‐L1 (ab205921; Abcam, Cambridge, UK), KRAS (12063‐1‐AP; Proteintech), HA (AB9110; Abcam) at a concentration of 1:5000, phospho‐AKT (Ser473, #9272; Cell Signaling Technology (CST), Danvers, MA, USA), total‐AKT (#4691; CST), phospho‐mTOR (Ser2448, #5536; CST), total‐mTOR (#2983; CST), phospho‐ERK (Thr202/Thr204, ab1812; Wanlei, China) at a total of 1000 (WL021, Wanlei, CST), phospho‐180 (Thr202/Thr204, IgG (WLP1512, Wanlei), with a total of 1000.

The 100 follicles were pooled from 5 mice at a time for each group. Total protein was extracted from PM and ES follicles using RIPA lysate (P0013B, Beyotime Biotech, Beijing, China) for Western blotting analysis. After SDS‐PAGE, the proteins were transferred to PVDF membrane, which was blocked with 5% skim milk for 1 hour at room temperature. Then, the membrane was incubated with antibodies to FSHR (bs‐20658R; Bioss), Ki‐67 (A2094; ABclonal), 3β‐hydroxysterol dehydrogenase (3β‐HSD) (bs‐3906R; Bioss), P450 17alpha‐hydroxylase (CYP17) (bs‐3853R; Bioss), P450 aromatase (CYP19) (bs‐0114R; Bioss), GAPDH (bs‐2188R; Bioss) or β‐actin (Ac026, ABclonal) overnight at 4°C. After washing, secondary antibody of HRP‐labeled goat anti‐rabbit antibody (bs‐0295G‐HRP; Bioss) was added and incubated for 1 hour at room temperature. Finally, the membrane was subjected to color development and photographed using a Tanon 4600 imaging system. Image was further analyzed by grayscale analysis using Tanon GIS ID software. The experiment for each group was repeated 5 times, and 5 mice were used each time.