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. Author manuscript; available in PMC: 2010 Apr 23.
Published in final edited form as: Mol Cell. 2009 Oct 23;36(2):315–325. doi: 10.1016/j.molcel.2009.09.037

Figure 4. Viral activation of IRF3 depends on catalysis by Ubc5.

Figure 4

(A) RNAi knockdown and expression rescue of Ubc5. U2OS cells stably expressing tetracycline-inducible shRNA against Ubc5b and Ubc5c together with Flag-tagged Ubc5c (wild-type or C85A) were treated with tetracycline (Tet; 1 μg/ml) for 7 days, then expression of Ubc5 was examined by immunoblotting with an antibody against all Ubc5 isoforms. (B) Active Ubc5 is required for IRF3 activation by Sendai virus (SeV). U2OS cells were treated with tetracycline as described in (A), then infected with SeV for the indicated time. Dimerization of endogenous IRF3 was analyzed by native gel electrophoresis. The asterisk (*) indicates a non-specific band cross-reacting with the IRF3 antibody. (C) Active Ubc5 is required for viral induction of IFNβ. U2OS cells were treated with tetracycline for 6 days and then infected with Sendai virus for 16 hours. IFNβ in the culture medium was measured by ELISA. The error bars represent variation ranges of duplicate experiments.