TRIM65 promotes K63-linked polyubiquitination of MDA5. (A) Immunoblot analysis of MDA5 ubiquitination in HEK-293T cells transfected with constructs encoding various combinations (above lanes) of Flag-MDA5, Flag–RIG-I, GFP-TRIM65, and HA-Ub, followed by immunoprecipitation (IP) with anti-Flag antibody. EV, empty vector. (B) In vitro ubiquitination assay for purified MDA5 by TRIM65. (C) Immunoblot analysis of MDA5 ubiquitination in HEK-293T cells transfected with constructs encoding various combinations of Flag-MDA5, GFP-TRIM65, GFP-TRIM65 without RING domain, and HA-Ub, followed by IP with anti-Flag antibody. (D) Immunoblot analysis of ubiquitination of MDA5 domains in HEK-293T cells transfected with constructs encoding various combinations of Flag-tagged MDA5 proteins, GFP-TRIM65, and HA-Ub, followed by IP with anti-Flag antibody. (E and F) Immunoblot analysis of ubiquitination of MDA5 helicase domain in HEK-293T cells transfected with constructs encoding various combinations of Flag-helicase, GFP-TRIM65, HA-Ub, HA-tagged mutant ubiquitin with substitution of alanine for all lysine residues except the residue noted (K48 and K63; E), or HA-tagged mutant ubiquitin containing a single lysine to arginine mutation at position 63 (K63R; F), followed by IP with anti-Flag antibody. (G) Immunoblot analysis of endogenous MDA5 ubiquitination in WT or Trim65^−/− BMDMs stimulated with EMCV-RNA for 3 h, followed by IP with anti-MDA5 antibody. Data are representative of three independent experiments.