Figure 1.

A, In vivo phosphorylation analysis. Plasmids encoding WT RB (lanes 1–4), 706F mutant RB (lanes 5–8), and the incomplete-penetrant mutant 712R (lanes 9–12) were cotransfected with the indicated cyclin plasmids (D2, D3, and E) into the RB (−/−) tumor cell line H2009 and were subjected to α-RB immunoblotting. Phosphorylation is indicated by the slight retardation in migration of the RB protein signal (ppRB). Protein-size marker is shown on the left. B, In vitro E2F1 binding assay. cDNA plasmids encoding WT or the 706F and 712R mutants were in-vitro translated with [³⁵S]-methionine and were subjected to SDS-PAGE analysis before (1 μl of lysate; lanes 1, 4, and 7) or after precipitation with either GST alone (10 μl of lysate; lanes 2, 5, and 8) or GST-E2F1 fusion proteins (10 μl of lysates; lanes 3, 6, and 9).