Fig 4.

Transcriptional activity of IRF7 mutants. Transactivation of the Gbp2 promoter by different mutants of IRF7. Stat1^−/− MEFs (D) or Tbk1/Ikbke^−/− MEFs (B) were transfected with WT IRF7 or the IRF7 mutants indicated (A). Fifty nanograms of IRF7 constructs were transfected alone or cotransfected with 1 μg TBK1 (B) as indicated. Gbp2-firefly luciferase reporter gene expression is indicated as fold induction relative to cells transfected only with reporter construct and normalization to cotransfected, constitutively expressed Renilla luciferase reporter. (C) Protein expression of IRF7 M1, M5, M15, and WT in Stat1^−/− MEFs was detected by Western blot analysis and probing the blot with anti-tag antibodies recognizing Flag-tagged IRF7 M1, M5, and M15 or HA-tagged WT IRF7. Pan-ERK levels were analyzed as a normalization control. (E) Irf7^−/− MEFs were transfected with 2 μg of a WT IRF7 construct, an empty vector control, or the IRF7 M1 mutant and treated for 6 h with IFN-γ (+) or left without treatment (−), followed by determination of SOCS1 mRNA expression. qPCR and luciferase measurements were made in triplicate. All experiments were repeated at least six times.