Fig. 1. Specificity of the antibody for tri-methylated lysine 20 of histone H4 (anti-Me(3)K20H4). (A) The anti-Me(3)K20H4 antibody recognises methylated H4 by western blotting. Histones from various sources were separated by SDS-PAGE, blotted and probed with anti-Me(3)K20H4. The sources of the histones were: turkey erythrocyte nuclei (lane 1), turkey erythrocyte core histones (lane 2), recombinant H4 (lane 3) and calf thymus core histones (lane 4). The bottom row shows a Coomassie Blue-stained profile of the samples used in the immunoblot. (B) Specificity of anti-Me(3)K20H4 antibody for the trimethylated form of K20 as determined by dot blotting. Serial dilutions (lanes left to right) of the non modified (I), mono- (II), di- (III) and tri-methylated (IV) K20 peptides were dot blotted onto nitrocellulose and then probed with anti-Me(3)K20H4. (C) The antibody is specific for Me(3)K20H4 in a competition assay using specific methylated peptides and membrane-bound histone H4. Calf thymus histone H4 is identified on an immunoblot by the anti-Me(3)K20H4 antibody alone (lane 1) or preincubated with non modified (lane 2), Me(1)K20H4 peptide (lane 3) and Me(2)K20H4 peptide (lane 4). Preincubation of the antibody with the Me(3)K20H4 peptide abolishes binding to H4 histone (lane 5). (D) The antibody is specific for Me(3)K20H4 in a competition assay using immunofluorescence (bottom row; top row are the corresponding DAPI profiles). From left to right: no competitor, non modified K20H4 peptide, Me(1)K20H4 peptide, Me(2)K20H4 peptide, Me(3)K20H4 peptide. In the last panel a Me(3)K9H3 was used as a competitor. Only the Me(3)K20H4 peptide competes. Scale bar: 5 µm.