Protein Extraction and Western Blotting

Total protein was extracted from pachytene spermatocytes and round spermatids from control and BEP-treated rats using a radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with proteinase inhibitor cocktail (Sigma) and phosphatase inhibitor (Active Motif) at a concentration of 10 μl/ml of RIPA. Samples were homogenized in RIPA (100 μl/1 × 10⁶ cells) by sonication at power 20 (Vibra-Cell; Sonics and Materials) for 3–5 sec on ice. Homogenates were spun down for 10 min at 15 700 × g at 4°C. The supernatant containing total protein was transferred into a new tube and protein concentrations were determined using the BioRad DC Protein Assay Kit II (Cat. no. 500-0112) according to the manufacturer's instructions (BioRad). Samples were resolved in SDS polyacrylamide (w/v) gradient (4%–12%) Bis-Tris gels (Invitrogen) at 200 V for 35 min using MES (2-[N-morpholino] ethanesulfonic acid) SDS running buffer (Invitrogen). Gels were transferred onto polyvinylidene fluoride membranes (GE Healthcare Life Bio-Sciences Inc.) using NuPAGE transfer buffer (Invitrogen) at 100 V for 1 h. Membranes were washed briefly with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and blocked with 5% nonfat milk in TBS-T. Proteins were detected using antibodies specific for H4K5ac, H4K8ac, H4K12ac, H3K9m, H2AK119u, H1T2, and tH2B, all diluted in 5% nonfat milk in TBS-T (Table 1). Blots stained for acetylated H4 and methylated H3 were stripped with Western Blot Stripping Buffer (ZmTech Scientifique) for 15 min (as suggested by company protocol), and restained with H4 and H3 antibodies, respectively.