FIG. 7.

Preferential phosphorylation of H3 at Ser-10 by Aurora kinases. (a) HEK293 cells were transfected with expression plasmids encoding Myc-tagged Aurora-A, Aurora-B, and Aurora-C kinases and the mutated isoforms Aur-A_D274N, Aur-B_K106R, and Aur-C_D184N. Protein extracts prepared from transfected cells were subjected to immunoprecipitation using the anti-Myc antibody. Immunoprecipitated complexes were analyzed by Western blotting to test the expression level of each kinase. (b and c) In vitro kinase assay performed on immunoprecipitated complexes using a histone mix as the substrate. Autoradiography (b, top panel) and Western blotting with anti-P.H3 antibody (c, bottom panel) are shown. The equal amounts of histones in each reaction were verified by Coomassie staining (middle panel). (d and e) In vitro kinase assay performed using the same samples described in panel b and GST-H3 and GSTH3-S10A as substrates; autoradiography (top panel) and Coomassie staining (bottom panel) are shown. The asterisks indicate the bands corresponding to the autophosphorylated Aurora kinases.