Fig. 3.

Regulation of eEF2 phosphorylation by dsDNA-sensing. (A) NHDFs were infected with HCMV (multiplicity of infection = 3 pfu per cell). At the indicated times, total protein was collected, fractionated by SDS/PAGE, and analyzed by immunoblotting using the indicated antibodies. (B) As in A. (C) NHDFs were mock-infected (uninfected) or infected with HCMV as in A. After 6 h, total RNA was isolated and RT-qPCR was performed using primers specific for IL6 mRNA (**P < 0.01 by Student’s t test). (D) As in A except cells were infected with HCMV or UV-inactivated HCMV and total protein harvested at 24 hpi. (E) NHDFs were transfected with no DNA or synthetic immunostimulatory dsDNA. At the specified times posttransfection, total protein was collected and immunoblotting was performed using the indicated antibodies. (F) NHDFs were transfected with nonsilencing siRNA (control), p65/RELA siRNA, or cGAS siRNA and transfected with no DNA or dsDNA. At 24 h posttreatment, total RNA was isolated and RT-qPCR analysis was performed for eEF2K mRNA. Error bars indicate SEM (*P ≤ 0.05; **P ≤ 0.01; and n.s., nonsignificant by Student’s t test). (G) NHDFs transduced with a doxycycline (dox)-inducible FLAG-eEF2K lentivirus were transfected with (+) or without (−) synthetic dsDNA and treated with dox as indicated. After 24 h, total protein was collected and analyzed by immunoblotting with the indicated antibodies.