Figure 3.

Licensing inhibition prevents Cdk2 phosphorylation at T160. (A) Whole cell lysates and anti-Cdk2 immunoprecipitates were probed for the CDK inhibitors, p21 and p27 by immunoblotting. (B) NHF1 cells were transfected with orc2 siRNA for a total of 120 hrs. BrdU incorporation was analyzed as in Figure 1D resulting in average (of two experiments) 29% S phase for control treated and 4.9% for orc2 siRNA treated cells. Whole cell lysates were probed for endogenous p21, Orc2 and tubulin by immunoblotting. (C) NHF1 cells were transfected with control or cdc6 siRNA for 72 h (lanes 2 and 3) or treated with 2 mM hydroxyurea for 24 h (lane 1). Whole cell extracts were immunoblotted with antibodies to detect p53 phosphorylated at S15, Chk2 phosphorylated at T68, Chk1 phosphorylated at S317, Cdk1 and Cdk2 phosphorylated at Y15 (the antibody recognizes both kinases), Cdc6 and tubulin as indicated. (D) Extracts were transfected as in (C) except, that whole cell extracts were probed for phospho-Cdk2 (T160), Cdc6 and tubulin. (E) WI-38 cells (diploid lung fibroblasts) were transfected with a total of 75 nM of control or cdc6 siRNA, incubated for 72 h and processed for flow cytometric analysis as in Figure 1D. The bar graph reports the average percentage S phase cells in three independent experiments (left). The remaining samples were subjected to immunoblot analysis, and a representative set of blots is shown (right). (F) NHF1 cells were transfected with a total of 75 nM of control or cdt1 siRNA and analyzed as in (E).