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. 2017 Feb;214(2):459–473. doi: 10.1084/jem.20160592

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© 2017 Lang et al.

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Figure 7. — TRIM65 ubiquitinates MDA5 at lysine 743. (A) Immunoblot analysis of ubiquitination of MDA5 helicase domain in HEK-293T cells transfected with constructs encoding various combinations of Flag-helicase proteins, GFP-TRIM65, and HA-Ub, followed by immunoprecipitation (IP) with anti-Flag antibody. Helicase-N, N terminus of helicase domain; Helicase-C, C terminus of helicase domain. (B and C) Immunoblot analysis of MDA5 ubiquitination in HEK-293T cells transfected with constructs encoding various combinations of GFP-TRIM65, HA-Ub, Flag-tagged WT MDA5, or mutants with substitution of arginine for lysine residues at different sites, followed by IP with anti-Flag antibody. (D) Luciferase activity (top) and immunoblot analysis (bottom) of lysates of HEK-293T cells transfected with luciferase reporter constructs driven by promoters of genes encoding IFN-β, plus Flag-tagged WT MDA5 or mutants. Data are representative of three independent experiments (A–C) or from three independent experiments with biological duplicates in each (D, mean and sem of n = 6). Statistics were analyzed via an unpaired Student’s t test: ***, P < 0.001.