DDR inhibition by E4orf4 is PP2A-dependent.

(A) L11 cells in which a PP2A-B55 shRNA can be transiently expressed following Dox induction were induced with Dox (+) or left untreated (-). 48 hrs later, the cells were transfected with an empty vector (-), or with a vector expressing shRNA-resistant PP2A-B55-HA (+). One day later, dl366* and dl366*+E4orf4 mutant viruses were used to infect the transfected cells, and some of the cells were left uninfected (Mock). Protein extracts were prepared 24 hrs post-infection and subjected to Western blot analysis with the indicated antibodies. Quantitation and normalization were performed as described in the legend to Fig 1. (B) HEK293-derived clone 13 cells containing Dox-inducible WT E4orf4 or clone 3 cells expressing an E4orf4 mutant unable to bind PP2A (R81F84A: E4orf4-mut) were induced with Dox for three hrs (+E4orf4)) or were left uninduced (-E4orf4), and were then treated with 4 μM hydroxyurea (HU) or with 25 ng per ml neocarzinostatin (NCS), or were left untreated (Control). Protein extracts were prepared after 3 hrs incubation with the drugs and subjected to Western blot analysis, using antibodies to phosphorylated and non-phosphorylated Chk1 and to E4orf4. Quantitation and normalization were performed as described above.