(A) Western blot analysis with H3K64ac antibody (left panel) on nuclear extracts from untreated (−) or Na-butyrate-treated (+) HeLa cells. Ponceau staining is shown as loading control (right panel). (B), (C), (D), and (E) Immuno-dot blots showing specific reactivity of the H3K64ac antibody. Aliquots of indicated pmoles of linear H3 peptides were spotted on the membrane. Of note, detection of peptides in (E) has been checked with H3K9ac, H3K18ac, and H3K27ac specific antibodies. (F) Peptide competition assay of H3K64ac immunoblot. H3K64ac antibody was pre-adsorbed with 50 pmoles/ml of indicated peptides. Ponceau staining is shown as loading control (bottom panel). (G) The H3K64ac antibody detects globular, tailless H3. Nucleosomes from NIH3T3 cells were incubated with no (−) or increasing amounts of trypsin to cleave the histone tails and immunoblotted as indicated for H3K9ac, H3K18ac, H3K27ac, H3K64ac and H3. The position of full-length and tailless H3 is indicated and loading was controlled by ponceau staining. (H) Peptide competition assay in immunofluorescence. MEFs were treated overnight with Na-butyrate (NaB) and then stained with H3K64ac antibody, pre-incubated with 100 pmoles/ml of either unmodified or acetylated K64 peptide. (I) Immunoblot analysis of H3K64 acetylation state in additional cell lines (Drosophila S2, mouse MEFs, human HeLa) with H3K64ac antibody. Inhibition of HDACs by Na-butyrate treatment increases acetylation levels. Ponceau staining is shown as loading control (bottom panel).