Figure 5.

Inhibition of cyclin E/cdk2 does not block S phase in the absence of Rb. (A) Inhibition of S phase induction by p21. Mouse embryo fibroblasts (MEF) from wild-type (Rb+/+) or knockout (Rb−/−) embryos were held at density arrest for 24 hr and infected with either Ad-Con (control virus with empty expression cassette) at a moi of 125 ffu per cell or Ad-p21 (moi of 100 or 500 ffu per cell as indicated) and then replated at subconfluent densities. Cells were labeled with BrdUrd from 12 to 24 hr postinfection, and BrdUrd was detected by using indirect immunofluorescence. At least 300 nuclei were scored for BrdUrd immunofluorescence. (B) Inhibition of cdk activity by p21. Cells treated as in A or cells density-arrested for 24 hr were lysed in IP buffer and immunoprecipitated with antibodies against either cyclin E, cyclin A, or cdk2. The kinase assays were performed by using glutathione S-transferase–Rb as the substrate.