Figure 4

ICP0 induces destabilization of IRF7. (A) 293T cells were transfected with 100ug FLAG-tagged IRF3 and increasing amounts of ICP0 as indicated (100 ng, 250 ng and 500 ng in lanes 3, 4 and 5 respectively) or an EV control. Cell lysates were western blotted using anti-FLAG antibody to detect any change in the expression of IRF3 in the presence of ICP0. Presence of ICP0 was detected by immunoblotting (upper panel). The β-actin served as a loading control (lower panel). (B,C) 293T cells were transfected with constructs expressing the empty vector (EV), ICP0 or with key regulators of type 1 interferon pathway (B) Myc-tagged IRF5 or (C) Flag-tagged IRF7. Eighteen-hour posttransfection cell extracts were western blotted using anti-Myc or anti-FLAG antibodies to detect any change in the expression of target proteins. Presence of ICP0 was detected by immunoblotting (upper panels). β-Actin served as a loading control (lower panels). (D) 293T cells were transfected with 100 μg FLAG-tagged IRF7 and increasing amounts of ICP0 as indicated (100 ng, 250 ng and 500 ng in lanes 3, 4 and 5 respectively) or an EV control. Cell lysates were western blotted using anti-FLAG antibody to detect any change in the expression of IRF7 in the presence of ICP0. Presence of ICP0 was detected by immunoblotting (upper panel). The β-actin served as a loading control (lower panel). Results are representative of three independent experiments. Densitometric analysis was performed and graphs represent changes in total IRF7 protein levels relative to β-actin (C,D). Results are expressed as the mean ± SD in each case and are representative of three independent experiments expressed as IRF7 fold expression over β-actin. *p < 0.05, **p < 0.01 as determined by Student t test.