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Fig. 2. In vitro phosphorylation of histone H3 S10 and S28 by MSK1. (A) [{gamma}32P]-ATP kinase assays were performed with acid extracts from quiescent C3H 10T{gamma} cells (lanes 1, 2), recombinant Drosophila H3 (lanes 3, 4), recombinant reconstituted Drosophila octamers (lanes 5, 6) or without substrate (lane 7) in the presence (lanes 2, 4, 6, 7) or absence (lanes 1, 3, 5) of recombinant MSK1 as described in Materials and Methods. Proteins were separated on 15% SDS-PAGE and Coomassie-stained (panel ii). Phosphorylated protein bands were detected by autoradiography (panel i). (B) Kinase assays were performed with acid extracts from quiescent C3H 10T{gamma} cells (lanes 1, 2), recombinant Drosophila H3 (lanes 3, 4), recombinant reconstituted Drosophila octamers (lanes 5, 6) or without substrate (lane 7) in the presence (lanes 2, 4, 6, 7) or absence (lanes 1, 3, 5) of recombinant MSK1 as described in Materials and Methods. Proteins were separated on 15% SDS-PAGE and transferred to a PVDF membrane for western blotting using anti-phosphoS10-H3 (panel i) or anti-phosphoS28-H3 (panel ii), or Coomassie-stained (panel iii). (C) Kinase assays were performed using either recombinant Drosophila H3 (lanes 1-4), or recombinant reconstituted Drosophila octamers (lanes 5-8). Aliquots were removed after 0, 15, 30 and 45 minutes. Proteins were run on 15% acid-urea gels. Gels were transferred to PVDF membrane for western blotting with anti-phosphoS28-H3 (panel i) or anti-phosphoS10-H3 (panel ii), or were Coomassie-stained (panel iii).





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