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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 3. UL97 directs E2F3b phosphorylation during HCMV infection.

FIGURE 3

(A) HFFs were transfected with either an empty vector (−) or expression vectors for Flag-tagged E2F3a (3a) or E2F3b (3b). At 48 hr post-transfection, cells were harvested and analyzed by western blot with Flag or E2F3 antibodies. (B) Serum-starved human fibroblasts (HFFs) were mock-infected or infected with wild-type HCMV (V) at an MOI of 3. At 24 hr post-infection (hpi) protein lysates were harvested and incubated with (+) or without (−) lambda protein phosphatase (l-PPase) prior to analysis by western blot with the indicated antibodies. Short: short-term electrophoresis in 10% gel. Long: long-term electrophoresis in 7.5% gel. *: non-phosphorylated or non-specific band. (C) Serum-starved HFFs were infected with HCMV as in panel B except maribavir (MBV) or DMSO was added as indicated. Lysates were analyzed by western blot with the indicated antibodies. M, mock infection. (D) Saos-2 cells were transfected with both expression plasmids for Flag-tagged E2F3b and Rb together with either an empty vector (−) or one expressing HA-tagged wild-type (WT), L1 motif mutant (L1) or a kinase deficient (KD) UL97. 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.