FIGURE 3:

Cdk1 phosphoepitopes rise rapidly during early mitosis. HeLa cells synchronized by double thymidine block were fixed 9 h after the release from the second thymidine block and immunolabeled with following antibodies: MPM2 (A), pS-Cdk (B), and pNucleolin (C). The fluorescence intensities were plotted according to mitotic stage. To assess the stage of mitosis precisely, cells were costained with antibody against Lamin B and with DNA dye. Integrated fluorescence intensity of a cell was measured at the brightest plane of a z series taken at 1-μm intervals. Each bar on the graphs represents an average of 15–30 cells for each stage. Error bars denote standard deviation. Representative images of each stage are shown in Supplemental Figure 2. (D) Diagram depicting relationship between Cdk substrate phosphorylation and irreversible mitotic entry. Cells become committed to forward mitotic progression in prometaphase, when Cdk substrates become phosphorylated.