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. Author manuscript; available in PMC: 2012 Feb 5.
Published in final edited form as: Cell. 2011 Jul 21;146(3):448–461. doi: 10.1016/j.cell.2011.06.041

Figure 6. RIG-I and K63-Ub4 induce MAVS aggregation and activation on the mitochondrial membrane.

Figure 6

(A) Full-length RIG-I was incubated with 5′-pppRNA and/or ubiquitin chains as indicated at 22°C for 10 min. The mixtures were then incubated with mitochondria from HEK293T cells for 30 min before mitochondrial extracts were analyzed by SDD-AGE and SDS-PAGE. 0.25 μg of ubiquitin chains or mono-Ub was analyzed by silver staining (right panel). (B) GST-RIG-I(N) was incubated with different ubiquitin chains at 22°C for 10 min in the presence or absence of ATP or EDTA as indicated. The mixture was then incubated with mitochondria from HEK293T cells for the indicated time, followed by analysis of the mitochondrial extracts using SDD-AGE and SDS-PAGE. (C) Similar to (B) except that varying amounts of GST-RIG-I(N) and K63-Ub4 were used, and their incubation time with mitochondria were kept at 30 min. (D) GST-RIG-I(N) was incubated with K63-Ub4 and then with mitochondria for the indicated time, followed by analysis of the mitochondrial extracts using SDD-AGE and SDS-PAGE. (E) GST-RIG-I(N) was incubated with K63-Ub4 before incubation with mitochondria in the presence or absence of DTT. The mitochondrial extracts were analyzed by SDD-AGE, SDS-PAGE and IRF3 dimerization assays. Mitochondria from Sendai virus-infected cells were used as a positive control. (F) Mitochondrial extracts from (E) were fractionated by sucrose gradient ultracentrifugation followed by SDS-PAGE and immunoblotting with a MAVS antibody.