Western Blot Analysis of Testis

Western blotting was performed as previously described [31 (link)]. The testes from the Lhda-cKO mice and the controls were harveste. Then, the enhanced cracking fluid (17C11B02, BOSTER, Wuhan, China) was used to lyse the testis, which was further centrifuged at 12,000× g at 4 °C for 30 min. Equal amounts of protein (45 μg) were loaded onto a 5–15% SDS-PAGE gel (Cat#P1200, Solarbio, Beijing, China), electrophoresed, and transferred to cellulose acetate membranes (0.45 µm), which were blocked with 5% nonfat blocking-grade milk. Primary antibodies (LDHA, 1:1000, AB101562, Cambridge UK; β-actin, 1:2000, GB11001, Servicebio, Wuhan, China) were incubated with the membranes overnight at 4 °C. The secondary antibody was used properly at room temperature for 2 h (goat to rabbit, 1:4000, BA1032, Boster, China; goat to mouse, 1:4000, BA1031, Boster, China). ECL Plus chemiluminescence reagent kit (AR1197, BOSTER, China) was used captured the bands, which were quantified with optical methods using ImageJ software (4200SF, Tanon, Shanghai, China). The results were normalized using β-actin as a control.