Bca quantification

Proteins were extracted from ovaries with RIPA Lysis Buffer (Beyotime, P0013C), and protein concentration was adjusted by BCA quantification (Beyotime, P0010S) and denatured at 95 °C for 10 min. Samples were separated on 10% polyacrylamide gels (20325ES62, YEASEN) and blotted on PVDF membranes (Immobilon-P, Millipore). Incubate in 5% skim milk for 1 h, then incubate with primary antibody overnight at 4 °C. Then TBST was washed 3 times for 5 min each, and the secondary antibody was incubated at room temperature for 2 h and washed again with TBST. The HyperSignal ECL chemiluminescence kit (4 A Biotech Co., Beijing.) was used to develop the color and the chemiluminescence signals were captured with a Tanon 4600 chemiluminescence imaging system (Tanon, Beijing, China). The Primary antibodies used in this study are presented in Table S2.

Cells were lysed with cell lysis buffer (Beyotime, Shanghai, China) prior to BCA quantification (Beyotime, China), and 30 μg of total protein was loaded and separated with a 10% SDS-PAGE gel. Then, the protein was transferred onto PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 5% skim milk, primary antibodies against ZNF385A (26288-1-AP) (1:2000, Proteintech, Rosemont, IL, USA), ZNF346 (20794-1-AP) (1:3000, Proteintech, Chicago, IL, USA), and GAPDH (10494-1-AP) (1:20,000, Proteintech, USA) were utilized to incubate the membranes throughout the night at 4 °C. The secondary antibody (A0208) (1:2000, Beyotime, China) was added the following day, and the protein bands were visualized with BeyoECL Plus (Beyotime, China).