Fig 3.

TBK1 is required for the regulation of IFN-γ-induced genes by IRF1 and IRF7. (A to D) Expression of GBP2, TAP1, SOCS1, and IRF1 mRNA in WT and Tbk1/Ikbke^−/− MEFs after IFN-γ treatment for the times indicated was analyzed by qPCR and normalized to GAPDH mRNA levels. (E) Stat1^−/− MEFs were transfected with either IRF1 or IRF7, and Gbp2-firefly luciferase reporter activity was measured. The values are expressed as fold induction relative to cells transfected only with reporter construct and normalization to a cotransfected, constitutively expressed, Renilla luciferase reporter. (F) Tbk1/Ikbke^−/− MEFs were transfected with IRF7 or TBK1 alone or with a combination of both. MEFs were treated with IFNAR1 blocking antibody for the whole period of transfection or left untreated. (G) Stat1^−/− MEFs were transfected with IRF7 or TBK1 alone or in combination. Transfected cells were stimulated overnight with IFN-γ or left untreated. (H) Stat1^−/− MEFs were transfected with either WT IRF7 or TBK1 or a combination of both. IRF7 amounts were left constant, whereas TBK1 amounts were varied as shown. (I) Stat1^−/− MEFs were transfected with IRF1 or TBK1 alone or in combination. In panels F to I, Gbp2 luciferase reporter activity was measured as described for panel A. (J) Requirement for TBK1/IKKε-mediated IRF7 phosphorylation determined by 2D gel electrophoresis. WT or Tbk1/Ikbke^−/− MEFs were treated with IFN-γ for 4 h or left untreated. Nuclear extracts were prepared and subjected to 2D gel electrophoresis. IRF7 isoforms were analyzed by Western blotting. qPCR and luciferase measurements were made in triplicate. All experiments were repeated at least three times.