FIG. 5.
Repression of E2F by partially phosphorylated pRb. (A) Repression of E2F transcriptional activity by p16INK4A and cdk2DN. Cells transfected with the indicated plasmid and either a wild-type (3× E2F; ▪) or a mutant (3× mut; □) E2 promoter-luciferase reporter construct were analyzed for luciferase activity. The mean and standard error of the mean from three independent experiments are shown. (B) Identification of E2F-associated pRb by immunoblot analysis of pRb from lysates prepared from asynchronously growing (lanes 1 and 3) or cdk2DN-transfected but not sorted (lanes 2 and 4) U2-OS cells before (lanes 1 and 2) or after precipitation with Sepharose 4B–GST-DP1-E2F1 (lanes 3 and 4) or Sepharose 4B-GST alone (lanes 6 and 7) or prepared from cdk2DN-transfected sorted U2-OS cells (lane 5).