Super ecl plus western blotting substrate

As described in a previous protocol (Zhang et al., 2022a (link)), proteins from HEK293T cells were extracted using RIPA (radioimmunoprecipitation assay) lysis buffer (Applygen, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitors on ice. For sperm samples, 1% SDS was added to RIPA lysis buffer. The supernatants were collected following centrifugation at 12,000 × g for 20 min. Proteins were electrophoresed in 10% SDS‒polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (GE Healthcare). The blots were blocked in 5% milk and incubated with primary antibodies overnight at 4°C, followed by incubation with anti-rabbit or anti-mouse IgG H&L (HRP (horseradish peroxidase)) (Abmart) at a 1/10,000 dilution for 1 hr. The signals were evaluated using Super ECL Plus Western Blotting Substrate (Applygen) and a Tanon5200 Multi chemiluminescence imaging system (Tanon, Shanghai, China). Antibodies used in this study are listed in Supplementary file 5.