Fig. 8. Histone H3 phosphorylation is not mediated by the MAPK pathway in mouse spermatocytes. (A) Immunokinase assay of p90Rsk2 activity using H3 as substrate. p90Rsk2 was immunoprecipitated as described in the legend to Fig. 4 from cells treated as indicated in the figure and in the text, and activity was assayed using 0.1 mg/ml H3 and 10 mM ATP as substrates. At the end of the incubation, proteins were separated on a 10% SDS-PAGE and analyzed by western blot using either anti-phospho-H3 to detect phosphorylation of Ser10 in H3, or anti-p90Rsk2 to verify that equal amounts of enzyme were used in the assay. (B) Western blot analysis of extracts (20 µg) of cells treated as indicated in the figure and in the text using either the anti-phosphoH3 antibody to detect phosphorylation of Ser10 in H3 in intact cells, or the anti-H3 antibody to quantify the amount of H3 in the samples.
