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.
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