Rabbit polyclonal anti histone h3 antibody

Whole cell lysate (WCL) and histone extract were prepared from cultured cardiomyocytes, cardiac fibroblasts, and mouse hearts, and Western blotting was carried out as previously described [20 (link)]. In short, after the cells were harvested, WCL was prepared on ice with cell lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2% Nonidet P40 (Sigma-Aldrich, St. Louis, MO, USA), 0.2 mM ethylenediaminetetraacetic acid) for 5 min. Histone fractions from primary cultured cardiomyocytes and the heart tissue of TAC mice were isolated by acid extraction [22 (link)]. SDS-PAGE was used to resolve 10 μg of each fraction. In the Western blotting assays, the primary antibodies used were mouse monoclonal anti-α-SMA antibody (Sigma-Aldrich), mouse monoclonal anti-β-actin antibody (Sigma-Aldrich), rabbit polyclonal anti-histone H3 antibody, and rabbit polyclonal anti-acetyl-histone H3 (Lys9) (Cell Signaling Technology, Danvers, MA, USA); the secondary antibodies used were anti-rabbit antibody (MBL, Aichi, Japan) and anti-mouse antibody (MBL). An Amersham Imager 680 (GE Healthcare, Chicago, IL, USA) was used to visualize the blots, and ImageJ software version 1.52a (NIH) was used for quantification.

Whole-cell extracts, nuclear extracts, histone extracts, and a sample from the in vitro HAT assay were prepared and Western blots were performed as described previously [26 (link)]. For Western blotting, rabbit polyclonal anti-acetyl-histone H3K9 antibody (#9649, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antihistone H3 antibody (#9715, Cell Signaling Technology, USA), rabbit polyclonal anti-RNA Polymerase II antibody (sc-47701, Santa Cruz, Dallas, TX, USA), goat polyclonal anti-Cdk9 antibody (C-20) (sc-484, Santa Cruz, USA), rabbit polyclonal anti-Cyclin T1 antibody (sc-10750, Santa Cruz, USA), mouse monoclonal anti-β-actin clone AC-15 IgG (A1978, Sigma-Aldrich, USA), goat antirabbit IgG–HRP antibody (MBL, Tokyo, Japan), and sheep antimouse IgG (GE Healthcare, Chicago, IL, USA) were used. The signals were detected by a C-DiGit Chemiluminescent Western Blot Scanner (LI-COR, Lincoln, NE, USA) and a LAS-1000 Plus luminescent image analyzer (Fujifilm, Tokyo, Japan) and quantified using Image Studio LITE software (LI-COR, USA).