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. Author manuscript; available in PMC: 2012 Dec 29.
Published in final edited form as: Immunity. 2012 Jun 14;36(6):959–973. doi: 10.1016/j.immuni.2012.03.022

Figure 2.

Figure 2

K63 polyubiquitin chains bind to and activate MDA5. A. GST or GST-MDA5(N) was incubated with polyUb chains containing K63, K48, or linear linkage, and then pulled down with glutathione beads followed by immunoblotting with indicated antibodies. Input represents 10% of polyubiquitin used for GST pulldown (GST-PD) experiments. B. Endogenous polyUb chains in HEK293T cells were isolated using the procedure shown in the diagram (left). After centrifugation, the supernatant containing the ubiquitin chains was incubated with GST-RIG-I(N) or GST-MDA5(N), which were then incubated with mitochondria (P5) and cytosol (S5) to measure IRF3 activation. C. Endogenous polyUb chains associated with RIG-I(N) or MDA5(N) were isolated as in B., and then incubated with GST-RIG-I(N) followed by IsoT or CYLD treatment, or in reverse order, before IRF3 dimerization assay. Parallel experiments were carried out using free K63 polyUb chains as controls (lanes 13–18). D. HEK293T cells stably expressing GFP (control) or MDA5 were transfected with indicated RNA. IFNβ induction was measured by quantitative PCR (qPCR). E. Similar to B and C, except that endogenous polyUb chains associated with MDA5 were isolated from EMCV RNA-transfected HEK293T cells stably expressing MDA5-Flag. The polyUb chains were incubated with GST-RIG-I(N) and IsoT or CYLD in the indicated order, and then with mitochondria (P5) and cytosol (S5) to measure IRF3 dimerization. F. U2OS integrated with tetracycline-inducible shRNA against Ubc13 (shUbc13) were treated with or without tetracycline (Tet). IFNβ induction by EMCV RNA was measured by qPCR. G. WT or Trim25-deficient (KO) MEF cells were transfected with indicated RNA. IFNβ was measured by qPCR. H & I. Similar to F., except that U2OS cells stably expressing tetracycline-inducible shRNA against ubiquitin (shUb, left), and those in which endogenous ubiquitin was replaced with K63R ubiquitin (shUb/K63R, right), were transfected with EMCV RNA, followed by measurement of IFNβ by qPCR. J. RNA transfection of U2OS cells was carried out as in H and I, and then mitochondria (P5) were isolated and incubated with cytosolic extracts (S5) to measure IRF3 dimerization. Aliquots of the mitochondrial extracts were immunoblotted with a MAVS antibody. Results shown are representatives of two experiments.