The E3 ubiquitin ligase TRIM25 catalyzes Lys 48-linked ubiquitination of MAVS. (A) The sequence of TRIM25 and the matching endogenous peptides (highlighted in yellow) were identified by mass spectrometry. (B) HEK293T cells were infected with SeV H4 in the presence of MG132 for 6 hr. Next, endogenous MAVS was immunoprecipitated from cell extracts; the presence of MAVS and TRIM25 was examined by immunoblotting. (C) HEK293T cells were transfected with TRIM25-V5 or control plasmid for 24 hr. Next, endogenous MAVS was immunoprecipitated in denaturing conditions from cell extracts with specific antibody; the presence of MAVS and its ubiquitination was examined by immunoblotting. (D) HEK293T cells were transfected with TRIM25-V5 or control plasmid, and 72 hr after transfection, Mfn1, MAVS, V5 (TRIM25) and Bcl-2 were analyzed in cell extracts by immunoblotting. Values represent the ratio of the larger MAVS isoform band normalized with respect to the loading control. (E) HEK293T cells were transfected with the indicated plasmids. Twenty-four hours after transfection, immunoprecipitation and immunoblot analysis were performed with the indicated antibodies (upper panel). Expression of the proteins was examined by immunoblots with the indicated antibodies (lower panel). (F) Experiment was performed as in E. Myc-MAVSmut: Myc-MAVS(K7R/K10R) mutant.