TRIM32 destabilizes TRIF independent of its E3 ligase activity.

<p>(A) Murine TRIM32 inhibited TLR3- and TRIF-mediated ISRE activation in mammalian overexpression system. HEK293 cells were transfected with the indicated adaptor plasmid and ISRE reporter (0.1 μg each) and an HA-tagged expression plasmid for murine TRIM32 (0.05 μg). Twenty hours after transfection, luciferase assays were performed. *, p<0.05. (B) Interaction of TRIM32 and TRIF. HEK293 cells were transfected with the indicated plasmids for 24 hours before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. (C) Endogenous association of TRIM32 with TRIF. MLFs were left untreated or treated with poly(I:C) (100 μg/ml) or LPS (100 ng/ml) for the indicated times, followed by endogenous coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (D) Effects of murine TRIM32 on levels of TRIF, TRAF3, TBK1 and IRF3. HEK293 cells were transfected with the indicated plasmids for 24 hours before immunoblotting analysis with the indicated antibodies. (E) Effects of TRIM32-deficiency on poly(I:C)-indiced degradation of TRIF. MLFs were left untreated or treated with poly(I:C) (50 μg/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. (F) Effects of murine TRIM32 and its enzyme-inactive mutants on the levels of TRIF. HEK293 cells were transfected with the indicated plasmids for 24 hours before immunoblotting analysis with the indicated antibodies. (G) Effects of murine TRIM32 on ubiquitination of TRIF. HEK293 cells were transfected with the indicated plasmids for twenty hours before immunoprecipitation and immunoblotting analysis with the indicated antibodies. (H) Analysis of poly(I:C)- or LPS-induced transcription of downstream genes in <i>TRIM32</i><sup>-/-</sup> MLFs reconstituted with an empty vector, TRIM32-Flag (WT) or TRIM32 (ΔRING)-Flag. The reconstituted MLFs were stimulated with poly(I:C) (50 μg/ml) or LPS (50 ng/ml) for the indicated times before qPCR experiments.</p>