Rabbit monoclonal anti histone h3 antibody

Histone extracts were prepared from neonatal rat cardiomyocytes, Dahl-rats, and α-MHC-p300 TG mice as described previously [66 (link)]. For Western blotting, rabbit monoclonal anti-histone-H3 antibody, rabbit polyclonal anti-acetyl-histone-H3 (K9) antibody, and rabbit polyclonal anti-acetyl-H3 (K122) antibody (Abcam, Cambridge, UK) were used. The signals were detected with a C-DiGit Chemiluminescent Western Blot Scanner (LI-COR, Lincoln, Nebraska) and a LAS-1000 Plus Luminescent Image Analyzer (Fujifilm, Tokyo, Japan) and quantified using Image Studio LITE software (LI-COR).

Mouse testicular tissue, epididymal mouse spermatozoa and ejaculated boar and human spermatozoa were washed two times with TBS, and the pellets were dissolved in RIPA lysis buffer^{28 (link)} with 100 mM DTT, enriched with Complete Mini Protease Inhibitor Cocktail (Roche, Switzerland) and incubated for 30 min on ice. Thereafter, samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–15% separating Mini-PROTEAN^® precast gel and blotted using a Trans-Blot Turbo Transfer System onto PVDF membranes (Bio-Rad Laboratories, France). The membranes were blocked in 5% BSA in TBS with 0.05% Tween-20 (TBS-T) for 60 min at room temperature. The membrane was incubated with primary antibodies as mentioned above and diluted 1:1000 in 1% BSA in TBS-T overnight at 4 °C. A rabbit monoclonal anti-histone H3 antibody (1:1000, Abcam, Cambridge) was used as the internal control. Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:15,000; Invitrogen, Carlsbad, CA, USA) was applied for 60 min at room temperature. Target proteins were visualized using ECL Select Western Blotting Detection Reagent^® (GE Healthcare Life Sciences, UK) and a ChemiDoc^® MP System (Bio-Rad).