Fig. 4. USP15 enhances TRIM25- and RIG-I–mediated signaling.

(A and B) HEK 293T cells were transfected with the pGK–β-gal plasmid [which constitutively expresses β-galactosidase (β-gal)] and either (A) IFN-β or (B) NF-κB reporter plasmids, together with plasmids encoding GST–RIG-I(2CARD) and TRIM25-V5 and increasing amounts of plasmid encoding USP15-Myc. Forty-eight hours later, luciferase and β-gal values were determined as described previously (16). Data are means ± SD of duplicate samples and are representative of four independent experiments. (C and D) IFN-β luciferase activity in HEK 293T cells that were transfected with the indicated constructs was determined as described in (A). Data are means ± SD of duplicate samples and are representative of (C) three or (D) two independent experiments. (E) Measurement of IFN-β luciferase activity in HEK 293T cells transfected with plasmid encoding GST–RIG-I 2CARD alone or together with plasmid en-coding TRIM25-FLAG and that stably expressed either nontargeting control shRNA (sh.C) or a USP15-specific shRNA (sh.USP15). Luciferase and β-gal values were determined 48 hours after transfection. Data are means ± SD of duplicate samples and are representative of three independent experiments. (F) Twenty-eight hours after transfection with the indicated siRNAs together with the IFN-β luciferase and pGK–β-gal plasmids, HEK 293T cells were mock-treated or were infected with SeV (5 HA U/ml). Luciferase and β-gal values were determined 20 hours later. Data are means ± SD from three independent experiments. **P < 0.01. (G) HEK 293T cells, stably expressing sh.USP15 or sh.C, were infected with SeV (50 HA U/ml) for 16 hours. The extent of ubiquitylation of endogenous RIG-I was then determined by immunoprecipitation with α–RIG-I antibody and Western blotting analysis with α-Ub antibody. Data are representative of three independent experiments.