Fig. 2.
mH2A1 is phosphorylated in the hinge region on S137. (a) MS analysis identified two phosphorylation sites in the basic hinge region of mH2A1, T128 and S137 (asterisks). Sequence shown represents the peptide generated by GluC endopeptidase digestion. Peptides with both forms of alternate splice selection containing either KK or KKK were identified (underlined). The ARKS motif conserved in H3 is shaded. ClustalW alignment of the phosphorylated residues of mH2A 1.1, 1.2, and 2 is shown below. Note that phosphorylation potential exists for all three isoforms in the case of T128 but only for mH2A1.1 and 1.2 at S137. (b) mH2AS137ph antibody is specific as demonstrated by peptide competitions. HEK293 histones were used for peptide competitions with S137ph antibody. Membrane was cut into strips, probed with the antibodies indicated, and reassembled; Prebl., prebleed serum; un, unmodified peptide; and ph, phosphorylated peptide. mH2A IB was used as a control for size (last lane). Amido black staining of histones is used as a loading control. (c) Separation of mH2A isoforms demonstrates that both mH2A1 splice isoforms, mH2A 1.1 and 1.2, are phosphorylated at S137 in adult female and male mouse liver. (d) (Left) mH2AS137ph is found in all cell types examined: HEK293, HeLa, LF2 ES cells, 3-day retinoic acid-differentiated LF2 cells, and mouse 3T3 cells. (Right) Both male and female ES cells contain this PTM.