Knockdown of E2F4 or p107 and p130 does not allow UL97-L1m to relieve Rb-mediated suppression of E2F-responsive transcription.
A, Saos-2 cells were transfected with an siRNA targeting E2F4 or a control siRNA for 24 h. Cells were then transfected with a luciferase reporter driven by the E2F1 promoter together with an empty vector (−) or an expression plasmid for Rb and the indicated allele of UL97. Lysates harvested 48 h after plasmid transfection were analyzed for luciferase activity (top) and protein expression with the indicated antibodies (bottom). Luciferase activity was normalized to total protein concentration and is presented relative to the activity of the reporter without Rb or UL97 (set at 100%). Error bars denote the S.D. *, p < 0.05; **, p < 0.01. B, luciferase and Western blot analyses were performed as in A except cells were transduced with lentiviruses expressing shRNAs targeting either GFP or p107 and p130. Luciferase activity is presented relative to the activity of the reporter without Rb or UL97 in each transduced cell (set at 100%). KD, kinase-deficient.