Figure 3A and B IFNβ1 luciferase assays (upper panels) in HEK293T (A) and HEK293T-IRF3 KO (B) cells transfected with the indicated plasmids or infected with VSV. Immunoblot analysis showed the indicated constructs in HEK293T cells (bottom panels). 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 one-way ANOVA Data are from three independent experiments Error bars mean ± SEM). C The mRNA levels of Ifnb1 were measured by qRT-PCR in WT (n=3) and Akt2 KO (n=3) PEMs with Irf3 knockdown for 48 hours and VSV treatment for 6 hours. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent experiments Error bars mean ± SEM). D WT and Akt2 KO PEMs were infected with or without VSV for 6 hours. Then, cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti-AKT2 antibody, followed by immunoblotting using anti-IRF3 antibody (left panel). Alternatively, cell lysates were prepared for immunoprecipitation using anti-IRF3 antibody, followed by immunoblotting using anti-AKT2 antibody (right panel) to detect the endogenous interaction between AKT2 and IRF3. Data information: * Data are representative of two or three independent biological replicates E Immunoblot analysis of GST-AKT2 and His-IRF3 interaction in a GST pull-down assay. Data information: * Data are representative of two or three independent biological replicates F Immunoassay of IRF3 in dimer or monomer form by native-gel and p-IRF3 (Ser396), p-TBK1 (Ser172), AKT2 and ACTIN by SDS-gel in WT and Akt2 KO PEMs with VSV stimulation for 6 hours. Data information: * Data are representative of two or three independent biological replicates G Immunofluorescent microscopic imaging for (left panel) and statistics analysis (right panel) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 hour post-VSV infection. IRF3 (red), Nuclei (Hoechst, green). The white arrows indicate the nuclei with IRF3 translocation. Mock, n=2; VSV, n=3. Bar, 20 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are representative of two or three independent biological replicates Error bars mean ± SEM). H Immunoassay of IRF3, AKT2, LaminB1 and GAPDH in the nuclear and cytoplasmic fractions from the WT and Akt2 KO PEMs after VSV treatment for 6 hours. Tubulin and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates I Immunoassay of IRF3, SP1 and Tubulin in the nuclear and cytoplasmic fractions of the HEK293T cells overexpressed with indicated constructs for 24 hours and stimulated with VSV for 6 hours. Tubulin and SP1 served as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates J IFNβ1 luciferases assays were performed in HEK293T cells transfected with AKT2 and 14-3-3ε followed by VSV treatment. 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 one-way ANOVA Data are from three independent experiments Error bars mean ± SEM). K qRT-PCR analysis for the Ifnb1 mRNA levels in VSV-stimulated WT and Akt2 KO PEMs with 14-3-3ε siRNA pretreatment. 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 two-way ANOVA test Data are from three independent experiments Error bars mean ± SEM).