Figure 2. CRL4^Cdt2 contributes to DNA damage-induced PCNA monoubiquitination.

(A) ES2 cells were transfected with the indicated siRNA. After 48 hours, cells were treated with 500 uM cisplatin for the indicated time. Cell lysates were analyzed by immunoblotting with anti-PCNA, anti-Cdt2, anti-Cul4, anti-DDB1, and anti-Rad18 antibodies. (B and C) 293T cells (B) and MCF7 cells (C) were transfected with si-Cdt2. After 48 hours, cells were irradiated with UV (50 J/m2) and harvested at the indicated time post-radiation. Cell lysates were analyzed as above. (D and E) DT40 cells or DT40 deficient of Rad18 (Rad18−/−) were treated with 50 uM cisplatin or 1 mM hydroxyurea for the indicated time and analyzed as above. (F) DT40^Rad18−/− cells were transfected with the indicated siRNA. Three days after transfection, cells were either left untreated or treated with 50 uM cisplatin or 1 mM hydroxyurea for 8 hours before harvesting. Lysates were immunoblotted with anti-PCNA antibody. (G) Total RNA was prepared from DT40^Rad18−/− cells with or without siRNA treatment. Using β-actin as a control, cdt2 and usp1 mRNA were analyzed by quantitative real-time PCR and expressed relative to the control si-GL2 sample. Mean ± S.D. of 3 experiments.