Ab184665

Ovarian tissues were homogenized in RIPA buffer (Thermo Scientific, Rockford, IL, USA) and centrifuged at 12,000 × g for 20 min. The protein content of samples was determined using a BCA protein assay kit (Bio-Rad Laboratories, Carlsbad, CA). Aliquots of each sample were separated via 12% SDS-PAGE and then transferred onto a nitrocellulose membrane, which was blocked with non-fat milk for 1 h. The membranes were probed overnight at 4°C with antibodies against the following proteins: HO-1 (ab68477; 1:2000; Abcam); NRF2 (ab92946; 1:3000; Abcam), TNF-α (SAB5700627; 1:1000; Sigma), IL-1β (AB1413-I; 1:4000; Sigma), IL-6 (SAB5700632; 1:2000; Sigma); MVH (ab13840; 1:3000; Abcam); OCT4 (ab184665; 1:2000; Abcam); Ki67 (ab16667; 1:4000; Abcam), PCNA (mAb2586; 1:3000; Cell Signaling Technology), ATM (ab199726; 1:2000; Abcam), RAD51 (ab176458; 1:3000; Abcam), and GAPDH (ab181602; 1:3000; Abcam). After washing with PBST buffer solution three times, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Beyotime Institute of Biotechnology) for 1 h at 25 °C, and the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific Inc.). The blots were scanned and normalized to GAPDH for quantification.

For IP, anti-OCT4 antibody (ab184665, Abcam, UK) was incubated with protein A/G beads (Millipore, USA) at room temperature for 1 h. Next, the supernatant of cell lysis was incubated with beads conjugated with anti-OCT4 at 4 °C overnight, followed by elution with loading buffer at 98 °C for 5 min, and then subjected to IB. IB was performed in accordance with our previous study (Zhu et al. 2020a (link)). The protein blots were visualized in ChemiDoc XRS (Bio-Rad, USA) using chemiluminescence (NCM Biotech; China). The details of primary antibodies and secondary antibodies were listed in Additional file 5: Table S5. The intensity of protein blots quantified by ImageJ software was normalized to that of GAPDH, which was expressed as the ratio of the control group.

Cells were washed twice with PBS on 96-well plate, fixed in 4% polyoxymethylene for 20 min at room temperature, permeabilized with 0.5% Triton X-100 for 15 min and then blocked with 1% BSA -TBST for 30 min. Cells were incubated with primary antibodies (OCT4, ab184665, Abcam, USA; MVH, ab270534, Abcam, USA) overnight at 4 °C. The fluorescence-labeled secondary antibody (A-11004, A32731, Thermo, USA) was incubated for 1 h at room temperature. Nuclei were stained by DAPI (D1306, Invitrogen, USA) for 10 min. Images were examined with an Olympus microscope(IX73, Olympus, Japan).