Fig. 3: IKKε v2 increases IRF7 phosphorylation and IRF7 translocation in the presence of ubiquitin.
From: IKKε isoform switching governs the immune response against EV71 infection
a, b IKKε v2 strongly phosphorylates IRF7 in the presence of ubiquitin. Flag-IRF7 and each V5-IKKε isoform were ectopically expressed in HEK293 cells in the presence or absence of ubiquitin. The lysates were subjected to immunoprecipitation with anti-Flag beads and the phosphorylated IRF7 was detected with anti-pSer antibody (a). Total lysates obtained from HEK293 cells ectopically expressing each V5-IKKε isoform, Flag-IRF7 and HA-ubi were subjected to immunoblot analysis. The phosphorylated IRF7 was detected by IRF7-phospho-Serine471/472 (IRF7-pS471/472) antibody (b). β-actin was served as an internal control. c IKKε v2 strongly phosphorylates IRF7 in EV71 infection. Flag-IRF7 and each V5-IKKε isoform were ectopically expressed in RD cells followed by EV71 infection. The Flag-IRF7 was immunoprecipitated with anti-Flag beads and the phosphorylation was detected by IRF7-pS471/472 antibody. The co-immunoprecipitated V5-IKKε isoform was analyzed by anti-V5 antibody. d, e IKKε v2 facilitates IRF7 translocation. HeLa cells were cotransfected with each Flag-IKKε isoform and V5-IRF7 in the presence or absence of HA-ubi. Nucleus and cytoplasm fractions obtained from HeLa cells were applied to immunoblot with anti-Flag and anti-V5 antibodies (d). Each Flag-IKKε isoform was transfected into HeLa cells along with HA-ubi. The cell lysates were adapted to nucleus and cytoplasm fractionation and immunoblot with anti-Flag and anti-IRF7 antibodies (e). H3 and α-tubulin were used as nuclear and cytosolic markers, respectively.
