2.6. Western blot analysis

The expressions of Bax protein in testis tissues were determined by Western blot analysis. Total protein was extracted from 100 mg testis tissue using 300 μl radio immunoprecipitation assay (RIPA) lysis buffer that contained 1 mM PMSF and protein phosphatase inhibitor complex (Biosino Bio‐Technology and Science Inc., Beijing, China). Total protein concentration was determined using BCA protein assays (Thermo Scientific Technologies, Wilmington, DE, USA). Protein samples (20 μg) were separated by 10% SDS‐PAGE. The PVDF membranes were then incubated with the indicated antibodies, and rabbit anti‐β‐actin (Bioss) and rabbit anti‐Bax (Sangon, Biotech, Shanghai, China) were used. Protein expressions were measured using a FluorChem M Fluorescent Imaging System (ProteinSimple, Santa Clara, CA, USA) and normalized to β‐actin expression.