FIG 8.

eIF4A3 impedes IRF3 binding onto ISRE and the interaction of IRF3 and CBP/p300. (A) Effect of eIF4A3 on IRF3 binding to ISRE. HEK293T cells were transfected with HA-IRF3 in the absence or presence of Flag-eIF4A3 and infected with SeV at 24 h posttransfection. At 12 hpi, cells were lysed, the biotinylated or unbiotinylated DNA probes were incubated with streptavidin beads for 2 h at 4°C, and then the probe-coated beads were incubated with cell lysates at 4°C overnight. The beads were washed five times with cold IP lysis buffer, and Western blot analysis was done to detect IRF3 and eIF4A3 by using an anti-HA and anti-Flag antibody. The band intensities were quantified, and the HA-IRF3 levels are shown below. (B) The interaction between IRF3 and CBP/p300 in the absence or presence of Flag-eIF4A3. HEK293T cells were transfected with the HA-IRF3 and Flag- eIF4A3 or its control expression vector (Flag). After 24 h, cells were infected with SeV for 12 h. A co-IP assay was carried out using anti-HA antibody to immunoprecipitate endogenous CBP. The band intensities were quantified, and relative precipitated CBP/HA-IRF3 ratios are shown.