Protein Extraction and Western Blotting

The GCs were harvested and washed once in phosphate-buffered saline (PBS), then lysed on ice for 30 min with radioimmunoprecipitation assay (RIPA) buffer (CST, 9806), and supplemented with 1% (v/v) protease inhibitor Cocktail (HY-K0010) and 1% (v/v) phosphatase inhibitors (Cocktail I, HY-K0021; Cocktail II, HY-K0022; and Cocktail III, HY-K0023), which were purchased from MCE (Shanghai, China). Western blotting was performed as described previously (Wu et al., 2019 (link); Yang et al., 2020a (link)). Protein concentrations were determined using a BCA protein assay kit (TransGen Biotech, Beijing, China). Equal amounts of proteins (15–50 μg/lane) were separated by SDS-PAGE (12% acrylamide running gel) and transferred to a nitrocellulose membrane (BioTrace^TM NT; Pall Corp., FL, United States). The following antibodies were used in this experiment: beta catenin (ab32572; Abcam), inhibin alpha (ab81234; Abcam), HSD17B1 (ab134193; Abcam), MIF (ab227073; Abcam), caspase6 (ab185645; Abcam), laminA/C (MA3-1000; Thermo), and p-laminA/C-S22 (13448; CST, Shanghai, China). The antibodies were diluted to the recommended ratio with Beyotime (P0256; Shanghai, China) diluent. The Western blotting images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).