FIGURE 4.

CDK1 activity is required for substrate reaccumulation in late S phase. A, HCT116 cells were synchronized in early S phase via double thymidine block and 5 h after release were treated with RO-3306 or DMSO (control). Cells were harvested at the indicated time points and analyzed for DNA content by flow cytometry. B, HCT116 cells were treated as in A, harvested at the indicated time points, and analyzed via immunoblotting. C, HCT116 cells were treated as in A. Soluble proteins were extracted, and bound proteins were fixed in early S phase (during the arrest) or in late S phase 7 h after release into fresh medium. Endogenous PCNA^DNA, SET8, and CDT1 were detected by immunostaining as in Fig. 1. Late S phase cells containing PCNA^DNA were scored for the presence of SET8 or CDT1 as an indicator of inactive CRL4^CDT2 (n ≥ 75 for each sample). Error bars, S.D.