Figure 6. Antibodies supposedly specific for H3K56ac cross-react against other acetylated lysine residues of histone H3.

(a) Immunoblot of core histones purified from control and butyrate-treated HeLa cells (NaBut), as well as WT and rtt109Δ yeast cells, probed with an antibody from Upstate/Millipore (07-677). (b) Immunoblot of core histones purified from control and butyrate-treated HeLa cells probed with Epitomics antibody 2134-1. Comparable amounts of histones from control and butyrate-treated cells were loaded as evidenced from the Coomassie Blue-stained gel. (c) Immunoblot of core histones purified from yeast cells lacking the H3K56 acetyltransferase Rtt109 (rtt109Δ) or WT cells. In panels (a) to (c), the “H3K???ac” label indicates that the immunoblotting signal could not be ascribed to a specific acetylated lysine(s). (d) Amino acid sequences flanking histone H3K56 and known acetylated lysines in the histone H3 N-terminal tail. Identical residues neighbouring acetylated lysines are highlighted. (e) Immunoblot of core histones purified from butyrate-treated HeLa cells were probed with monoclonal antibodies from Epitomics (2134-1) or Active Motif (61061) that were pre-incubated with different competitor peptides acetylated at specific lysine residues (histone H3 acetylated peptides). Ø: No competitor peptide. Equal loading of histones from HeLa cells was verified either by stripping the blot and reprobing it with an antibody that detects unmodified histone H3 (top panel) or by Ponceau S staining (bottom panel). (*) In addition to histone H3, the Active Motif antibody recognized a band that migrated faster than H3. Detection of this faster migrating species was blocked by H3K9ac and H3K56ac competitor peptides, suggesting that it is related to histone H3. In the same histone sample preparation, the Epitomics antibody did not recognize the band that migrated faster than H3.