Anti histone h3

Western blot was performed using testicular tissue as described in our previous study [33 (link)]. The primary antibodies included anti-activating transcription factor 4 (ATF4, Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti-Bcl-2-associated X protein (Bax, Cell Signaling Technology, 1 : 1000), anti-B-cell lymphoma 2 (Bcl-2, Santa Cruz Biotechnology, 1 : 2000), anti-binding immunoglobulin protein (BIP, Cell Signaling Technology, 1 : 1000), anti-caspase12 (Cell Signaling Technology, 1 : 1000), anti-cleaved caspase3 (c-caspase3, Cell Signaling Technology, 1 : 1000), anti-C/EBP homologous protein (CHOP, Cell Signaling Technology, 1 : 1000), anti-GAPDH (Santa Cruz Biotechnology, 1 : 2000), anti-histone H3 (Santa Cruz Biotechnology; 1 : 1000), anti-intercellular adhesion molecule 1 (ICAM-1, Santa Cruz Biotechnology, 1 : 500), anti-inducible nitric oxide synthase (iNOS, Cell signaling Technology, 1 : 1000), anti-NRF2 (Santa Cruz Biotechnology, 1 : 1000), anti-pro-caspase3 (Cell Signaling Technology, 1 : 1000), and anti-vascular cell adhesion molecule 1 (VCAM-1, Santa Cruz Biotechnology, 1 : 500).

For preparation of soluble protein in cell lysates, cells were first washed twice with ice-cold PBS and sonicated in 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% (w/v), Nonidet P-40 with protease(s) and 25 mM NaF, 2 mM Na₃VO₄, 0.1 mM PMSF, and 20 μg/mL aprotinin. The soluble and insoluble fractions were prepared by centrifugation at 15,000 × g for 15 min at 4°C. The soluble proteins were separated by electrophoresis through SDS-polyacrylamide gels (6, 8, and 15% w/v acrylamide) and then electrophoretically transferred to a PVDF membrane. [Subsequently, each membrane was blocked with 5% (w/v) skim milk in Tris-buffered saline containing 0.1% (w/v), Tween-20 for 1 h at room temperature and then hybridized with the appropriate primary antibody (diluted in tris-buffered saline) with gentle agitation overnight at 4°C. After washing three times with this buffer, each membrane was incubated with an appropriate secondary antibody for 1 h at room temperature. Each immunopositive band was visualized with enhanced chemiluminescence detection (GE Healthcare). The following antibodies and chemicals were used: anti-EZH2 (1:1000; BD), anti-H3K27me3 (1:1000; Abcam), anti-histone H3 (1:1000; Santa Cruz Biotechnology), and anti-α-tubulin (1:5000; Sigma); MG-132 was from Millipore, and 3-deazaneplanocin A-HCl was from Cayman Chemical Company. Images were quantified with Image J software.

Fresh tissues and cells were lysed with cell lysis buffer (Beyotime Biotechnology) and western blot was performed as described previously [18 (link)]. Briefly, 40 μg total proteins were applied to separation with SDS–PAGE gel. After the electrophoresis, the proteins were transferred to PVDF membranes (Millipore), followed by blocking in the TBST buffer containing 5% fat-free milk. The membranes were then incubated with indicated antibodies overnight at 4 °C, and then washed and incubated with HRP-conjugated secondary antibodies (Zhongsanjinqiao) for 2 h at room temperature and finally visualized using Chemiluminescent ECL reagent (Vigorous Biotechnology). The following antibodies were used in this work: Anti-GAPDH (Cell Signaling Technology), anti-JMJD2A (Cell Signaling Technology), anti-Histone H3 (Santa Cruz Biotechnology), anti-H3K9me3 (Abcam), anti-H3K36me3 (Abcam), anti-mTOR (Cell Signaling Technology), anti-p-mTOR (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-p-Akt Thr308 (Cell Signaling Technology), anti-S6K1 (Cell Signaling Technology), anti-p-S6K1 (Cell Signaling Technology).