Histone Acetylation Assay Protocol

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).