FIGURE 3.

Phosphorylation at Ser²⁸⁶ and Ser³⁰¹ amplifies the Cdk1 activation loop and then promotes mitotic entry. After the release of the second thymidine block, experiments were performed in the presence (C) or absence (A and B) of nocodazole as described under “Experimental Procedures” (see supplemental Fig. S2D). Graph data of the (cumulative) mitotic index at each time point indicate the mean ± S.E. for 200 cells from three independent experiments (A and C). *, 0.01 < p < 0.05; **, p < 0.01 (A and C). To evaluate the expression level of each exogenous Myc-Chk1 protein, samples were immunoblotted with anti-Chk1 antibody (A and C). Cyclin B₁-Cdk1 complexes were purified from cell extracts as anti-cyclin B₁ or anti-Cdk1 immunocomplexes, and in vitro kinase assays of activity toward histone H1 were performed. Activity was visualized by autoradiography of H1 (B, first and second rows). To further evaluate Cdk1 kinase activity, each sample was also subjected to immunoblotting with the indicated antibodies (B, lower rows). IP, immunoprecipitation.