Figure 2.

Overexpression of cyclin E/cdk2 induces Rb phosphorylation and E2F-dependent transcription. (A) Assay for Rb phosphorylation. Cells treated as in Fig. 1 were harvested in SDS/PAGE loading buffer at 24 hr postinfection, and Rb (Endog. Rb-P) was detected by using Western blotting (Upper). Alternatively, cells treated as in Fig. 1 were lysed in immunoprecipitation buffer, and the lysates were used for immunoprecipitation with antibodies specific for cyclin E, and the cyclin E-associated kinase assays were performed as described (42) by using histone H1 as a substrate (Lower). (B) E2F activity measured by using Northern analysis. Cells treated as in A were harvested at 24 hr postinfection and processed for Northern analysis by using specific probes for proliferating cell nuclear antigen (PCNA), B-Myb, and glyceraldehyde-3-phosphate dehydrogenase.