Wip lysis solution

Cultured mouse ovaries were lysed in WIP lysis solution (Cell Signaling Technologies, USA). Sample proteins were separated by electrophoresis by 10% SDS‐PAGE and transferred to PVDF (polyvinylidene fluoride) membranes (IPVH00010, Millipore, USA). Then, the membranes were blocked with 5% nonfat‐dry milk for 60 minutes and incubated at 4°C overnight with the following primary antibodies. The primary antibodies were as follows: p‐FOXO3a (9466, 97 kDa, 1:1000, Cell Signaling Technologies, USA), FOXO3a (12829, 82‐97 kDa, 1:1000, Cell Signaling Technologies, USA), p‐AKT (4060, 56 kDa, 1:1000, Cell Signaling Technologies, USA) and AKT (4691, 56 kDa, 1:1000, Cell Signaling Technologies, USA). The membranes were washed thoroughly with tris‐buffered saline with tween and incubated with the appropriate secondary antibody (1:5000, ZSGB‐BIO, China). The level of β‐actin (42 kDa, 1:1000, Sigma, USA) was used as an internal control. The membranes were visualized by the SuperSignal detection system (Prod 34080, Thermo, USA). To quantify the results of immunoblot, the image was quantified using Image J software.

The GCs in different groups were collected and then lysed in WIP lysis solution (Cell signaling technology, United States). Separated the prepared protein samples by electrophoresis with 10% SDS-PAGE and transferred the protein to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, United States). Then, blocked the membranes by using 5% nonfat-dry milk for 1 h at 37°C and incubated the membranes at 4°C overnight with the primary antibodies to be tested. The used primary antibody in the western blot was H2AK119ub1 (D27C4, rabbit, 1:1000, Cell signaling technology, United States). After overnight incubating, the membrane was washed with tris-buffered saline with 0.05% tween (TBST) for 30 min and then processed to incubate with the secondary antibody at room temperature for 1 h (1:5000, Beyotime, China). The level of α-Tubulin (A0208, rabbit, 1:1000, Beyotime, China) was used as an internal control. The membrane was washed with TBST for 30 min. The protein on membrane was captured by the Super Signal detection system (Prod 34080, ThermoFisher Scientific, United States). The images were then quantified by Image J software.