Figure 4A Immunoblot analysis of IRF3 in the cytoplasmic and nucleic fractions of HEK293T-IRF3 KO cells with overexpression of IRF3 and AKT2 or AKT2-S309A/T474A. Data information: * Data are representative of three independent experiments B Immunoblot analysis of in vitro kinase assay by the phosphorylation-gel. HA-AKT2 was immunoprecipitated from HEK293T cells by anti-HA/IgG antibody and Protein-G beads, and His-IRF3 was purified from E.coli cells by anti-His agarose beads. His-IRF3 alone and anti-IgG-beads added with His-IRF3 served as control. The arrow indicated the phosphorylated IRF3. Data information: * Data are representative of three independent experiments C IFNβ1 luciferase assays and immunoblot analysis showed the HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 hours and VSV infection for 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a two-way ANOVA test Data are from at least three independent experiments Error bars mean ± SEM D Immunofluorescence microscopy (top panel) of AKT2-, Flag-IRF3- or Flag-IRF3-T207A-overexpressed MEF cells after poly(A:T) stimulation for 6 hours. Flag (IRF3, red) and Hoechst (nuclei, blue). Immunoassay of nuclear-cytoplasm extractions (bottom panel) from HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours. GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively. Bar, 20 μm. Data information: * Data are representative of three independent experiments E HEK293T-IRF3 KO cells were overexpressed with indicated plasmids, and the cell lysate was conducted to immunoblot analysis by phosphorylation-gel for the assay of phosphorylated IRF3 (the upper band) by anti-Flag antibody. Data information: * Data are representative of three independent experiments F Survival rates (left panel) until 72 hours and H&E staining (right panel) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments G qRT-PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later. Every dot represents three zebrafish embryos. Horizontal vertical square bracket shows the statistical analysis of comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=15; PBS VSV, n=9; IRF3 VSV, n=16; IRF3-T207A VSV, n=11; AKT2 VSV, n=14; AKT2-T309A/S474A VSV, n=9; IRF3 + AKT2 VSV, n=9; IRF3 + AKT2-T309A/S474A VSV, n=13; IRF3-T207A + AKT2 VSV, n=10. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are representative of three independent experiments Error bars mean ± SD). H The survival rates of over-expressed zebrafish larvae as long as 72 hours after the VSV challenge. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments