A19700

Ovarian granulosa cells were harvested, collected into cold EP tubers, resuspended in lysis buffer, centrifuged, and the supernatants were collected. The concentration of protein was determined by a BCA protein assay kit (KeyGen Biotech, Nanjing, Jiangsu, China). After protein denaturation, a total of 20 μg of protein was electrophoresed in SDS-PAGE. Next, the proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes. After transfer and blockage with 10% nonfat dry milk solution for 1 h, the PVDF membranes were incubated overnight with primary antibodies for cleaved caspase-3 (9664; 1:1000 dilution, Cell Signaling Technology (CST), Danvers, MA, USA), BCL-2 (ab182858; 1:1000 dilution, Abcam), BAX (ab32503; 1:1000 dilution, Abcam), CTSD (ab75852; 1:1000 dilution, Abcam), SQSTM1/P62 (A19700; 1:500 dilution, ABclonal, Wuhan, Hubei, China), BECLIN-1 (A11761; 1:500 dilution, ABclonal), LC3 (L7543; 1:1000 dilution, Sigma-Aldrich), and β-actin (AC026; 1:2000 dilution, ABclonal) at 4 °C, respectively. After washing with TBST, the membranes were no longer present as demonstrated upon incubation with secondary HRP-conjugated goat anti-rabbit (7074; 1:5000 dilution, CST) and anti-mouse (7076; 1:5000 dilution, CST) antibodies, respectively. Protein blots were scanned using a ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA).

Cells were lysed in RIPA buffer (P0013B, Beyotime, China) containing the complete cocktail of protease inhibitors (#11836153001, Roche, Switzerland). Protein concentrations were determined with the BCA protein assay kit (P0011, Beyotime, China). Proteins were separated by 4-20% SDS-PAGE, transferred to PVDF film and blotted with antibodies at 4 °C overnight. Secondary antibodies were pre-labelled at room temperature for 1 h. The PVDF film with the target protein was exposed in the visualizer (AI800, GE, USA). Antibodies were purchased from the following: ALKBH5 (ab195377, 1:1000, Abcam), Caspase-3 (A19654, 1:1000, Abclonal), SQSTM1/p62 (A19700, 1:1000, Abclonal), GAPDH (A19056, 1:10000, Abclonal).