Figure 3

The effects of Cdk2/cyclin E complex down regulation on the G1 checkpoint inE15.5 embryonic liver cells. Pregnant mice were subjected to 0 or 6 Gy IR at E11.5, E13.5, E15.5 and P0. At the indicated time points after IR, embryonic liver cells were isolated for WB. (A) Protein levels of the cyclins D1, A, E, and B1. (B) Protein levels of Cdk1 and Cdk2. In response to IR, Cdk2 expression was down-regulated at E13.5 and E15.5. (C) Protein levels of p21 and p27. In response to IR, a dramatic induction of p21 was observed only at E13.5/E15.5. (D) The lysates from embryonic liver tissues (E11.5 and E15.5) harvested at 0 and 16 hours after IR were immunoprecipitated (IPed) with an antibody against cyclin E, and bound Cdk2 was detected by WB (left panel). The interaction pattern was representative of 2–3 experiments. To confirm the reduced expression of Cdk2/cyclin E complex, the lysates were IPed with an anti-Cdk2 antibody and then immunoblotted (IB) for cyclin E. The same lysates were IPed with anti-Cdk2 antibody and IBed with total Cdk2 and phospho-Cdk2 (Thr 160) (lower panel). (E) The lysates were IPed with antibodies against cyclin A and B1, and bound Cdk1 and Cdk2 were tested by IB. (F) IR-induced p21 expression was significantly enhanced in E15.5 liver cells compared to E11.5 cells.