TRIP negatively regulates TLR3/4- and RIG-I–induced IFN-β production. (A and B) RT-PCR and Western blot analysis of TRIP expression in mouse peritoneal macrophages stimulated with LPS for indicated time periods. (C) Western blot analysis of TRIP expression in mouse peritoneal macrophages transfected with control siRNA or TRIP siRNA 1 or 2 for 36 h. (D) Mouse peritoneal macrophages were treated as in C and then stimulated with LPS or poly(I:C) for 6 h. ELISA analysis of IFN-β in the supernatants and quantitative RT-PCR analysis of IFN-β mRNA in peritoneal macrophages. (E) Mouse peritoneal macrophages were transfected with IFN-β reporter plasmid together with TRIP expression plasmid or control plasmid, and luciferase activity was analyzed after treatment with LPS or poly(I:C) for 6 h. (F) HEK293-TLR3 cells were treated as in E. (G) HEK293 cells treated with the indicated plasmids were transfected with poly(I:C) for the indicated time periods. IFN-β, TNF, and TRIP mRNA expression were analyzed by RT-PCR. (H) RAW264.7 cells were transfected with iNOS, GAS, or IFN-β reporter plasmid together with TRIP expression plasmid or control plasmid, and luciferase activity was analyzed after treatment as indicated for 6 h. **, P < 0.01; *, P < 0.05. Data are representative of three experiments (mean and SD of six samples in D, E, F, and I). Similar results were obtained in at least three independent experiments in A, B, C, and G.