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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: Virology. 2017 Dec;512:95–103. doi: 10.1016/j.virol.2017.09.009

FIGURE 6. Cancer associated R37Q mutation prevents E2F3b phosphorylation by UL97 or cellular cyclin E/CDK2.

FIGURE 6

(A) Saos-2 cells were transfected with a luciferase reporter driven by the E2F1 promoter together with either an empty vector (−) or different amounts (0.1, 0.2, or 0.5 μg/35-cm2 area) of expression plasmids for DP1 or the indicated allele of Flag-tagged E2F3b. WT, wild-type; DA, T27A/S38A; 27E, T27E; 38D, S38D; ED, T27E/S38D. Lysates harvested 48 hr after 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 E2F3b or DP1 (set at 1). Error bars denote the standard deviation of more than 3 replicates. Statistical analysis utilized a two-tailed unpaired Student’s t test. n.s., not significant. (B) Saos-2 cells were transfected with expression plasmids for Flag-tagged alleles of wild-type (WT) E2F3b or E2F3b with single amino acid mutation (RQ, R37Q; 27, T27A; 38, S38A) and either an empty vector (−) or one expressing HA-tagged wild-type UL97 (97) or cyclin E1/CDK2 (E/2). Lysates harvested 48 hr after transfection were analyzed by western blot with the indicated antibodies. The phosphorylation-dependent band shifts of E2F3b were detected in phosphate affinity (phos-tag) gels.