FIGURE 3.

Rereplication-induced human Cdt1 degradation requires DDB1 and the PIP motif of Cdt1. A, HCT116 cells were transfected with DDB1 siRNA for 72 h and geminin siRNA for 48 h as indicated. Proteins in whole cell lysates were detected by immunoblotting with the indicated antibodies. B, schematic diagram of full-length Cdt1 and derivatives used in this study. C, HCT116 cells were infected with adenovirus expressing PIPm Cdt1-V5 or control virus at an m.o.i of 150. 48 h after infection, cells were harvested and probed for ectopic Cdt1 with anti-V5 antibody or for rereplication by flow cytometry. D, HCT116 cells were transfected with plasmids encoding C-terminally RFP-tagged Cdt1, Cdt1-RFP PIPm or control vectors. 24 h after transfection, cells were irradiated as in 1F and collected 30-min post-irradiation. Endogenous and Cdt1-RFP were detected by immunoblotting with anti-Cdt1 antibody. E, HCT116 cells were infected with adenovirus expressing WT Cdt1-V5 or PIPm Cdt1-V5 at an m.o.i. of 40 4 h prior to transfection with siRNA targeting geminin or control sequence. Cells were collected 32 h after transfection and assayed by immunoblotting with anti-V5, geminin, and tubulin antibodies.