FIG 5.

FAF1 operates at or downstream of IRF3. (A) HeLa cells were transfected with IFN-β-Luc, a beta-Gal reporter plasmid, Flag-FAF1, or a control plasmid and Flag-RIG-I N, Flag-MAVS, Flag-TBK1, or a control plasmid (Vec). After 24 h, cells were collected, and relative luciferase activities were measured. The data represent the means ± standard deviations of three experiments. Overexpressed proteins are shown by Western blotting, with tubulin bands representing loading controls. (B and C) HeLa cells were transfected with IFN-β-Luc (B) ISRE-Luc (C), beta-Gal, Flag-FAF1, or a control plasmid and IRF3-WT-GFP, IRF3-5D-GFP, or a control plasmid. At 24 h posttransfection, relative luciferase activities were determined as described for panel A. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (for differences between Flag and Flag-FAF1 values in panels A to C). (D) HeLa cells were transfected with a control siRNA or FAF1 siRNA 2. At 48 h posttransfection, cells were cotransfected with Flag-RIG-I N, Flag-MAVS, Flag-TBK1, Flag-IKKε, IRF3-5D, or a control plasmid and IFN-β-Luc together with a beta-Gal reporter plasmid. At 24 h posttransfection, relative luciferase activities were determined as described for panel A. **, P < 0.01; ***, P < 0.001 (for differences between values for the control siRNA and FAF1 siRNA). (E) HeLa cells were transfected with IFN-α4-Luc, beta-Gal, Flag-FAF1, or a control plasmid and the indicated amount of IRF3-5D-GFP. At 24 h posttransfection, relative luciferase activities were determined as described for panel A. (F) HeLa cells were transfected with IFN-β-Luc, beta-Gal, Flag-FAF1, or a control plasmid and TRIF. At 24 h posttransfection, relative luciferase activities were determined as described for panel A. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (for differences between values for Flag and Flag-FAF1 in panels E and F).