Western Blot Protein Analysis of Ovaries

For each sample, proteins were extracted from 3 to 4 ovaries using the RIPA lysis solution (Beyotime, P0013C) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. They were then transferred onto a polyvinylidene fluoride membrane (immobilon-P^SQ transfer membranes, Millipore, ISEQ00010, USA). After blocking in TBST buffer (TBS with Tween-20) containing 5% bovine serum albumin, the membrane was incubated in primary antibody (Table S2) overnight at 4 °C, washed and incubated with HRP-conjugated goat anti-rabbit (Beyotime, A0208) or anti-mouse IgG (Beyotime, A0216) diluted in TBST at room temperature for 1.5 h. Ultimately, the BeyoECL Plus Kit (Beyotime, A0018) was used for chemiluminescence; β-ACTIN was used as housekeeping protein as previous reported^{21 (link)}.