H1.2K187 methylation is cell cycle-independent and HP1 binds only to methylated H1.4K26. (a) H1.2K187me is cell cycle-independent. Quantitative MS analysis of H1 extracted from unsynchronized HEK293 cells and synchronized cells in G1 and M phase. A selected ion monitoring experiment was used on the ion 414.745 m/z (corresponding to H1K187 methylation) to monitor the levels of methylation. Normalisation was done relative to unsynchronized cells. (b) Recombinant HP1 binds to H1.4K26me2 but not to H1.2K187me2. Peptide affinity purification of recombinant HP1β and γ (GST fusion protein). The upper panels show the anti GST WB of the elution and lower panels the Coomassie staining of eluted fraction. (c) Endogenous HP1 binds to H1.4K26me2 but not H1.2K187me2. Western Blot analysis of peptide affinity purification of nuclear extracts (HeLa extracts) with HP1α/β specific antibody. (d) Glp1 does not bind to H1.4K26me2 or H1.2K187me2. Western Blot analysis of peptide affinity purification of nuclear extracts (HeLa extracts) with Glp1 specific antibody. (e) JMJD2D demethylates H1.4K26me2 only. Methylation assay with G9a and Glp1 after demethylation of peptides by JMJD2D (Rad).