Fig. 3. Deacetylation of a synthetic phosphodiacetyl histone H3 peptide restores anti-phospho-H3 antibody binding. (A) Schematic summary of the phospho- (serine 10), diacetyl- (lysines 9 and 14) and phosphodiacetyl-H3 peptides synthesized for peptide screening and subsequent antibody generation. (B) The indicated mass of synthetic peptide was spotted onto Hybond-C in a volume of 1 µl. These correspond to histone H3 (lane 1), phospho-H3 (lane 2), diacetyl-H3 (lane 3) and phosphodiacetyl-H3 (lane 4). In vitro deacetylation of the synthetic phosphodiacetyl-H3 peptide was performed using recombinant yeast histone deacetylase HOS3. These peptide and enzyme samples were also spotted onto Hybond-C at the indicated mass in a volume of 1 µl. These correspond to HOS3 alone (lane 5), phosphodiacetyl-H3 plus HOS3 (lane 6) and phosphodiacetyl-H3 plus boiled HOS3 (lane 7). Spotted peptide and enzyme samples were air-dried and western analysis was performed using anti-phospho-H3 antibody.