(A) Diagram of differential centrifugation of cell homogenates. HEK293T cells infected with Sendai virus (+SeV) or mock treated (−SeV) were homogenized in hypotonic buffer, followed by sequential centrifugation to separate crude mitochondria (P5) from cytosolic supernatant (S5 and S100). (B) IRF3 and NF-κB activation in vitro. Mitochondrial fraction (P5) from Sendai virus-infected HEK293T cells or purified His6-tagged MAVS without transmembrane domain (His6-MAVSΔTM) was incubated with cytosolic extract (S5) from uninfected cells in the presence of ATP and 35S-IRF3. Dimerization of IRF3 was analyzed by native gel electrophoresis, followed by autoradiography. IκBα phosphorylation was analyzed by immunoblotting. (C) NEMO-interacting complex is required for IRF3 activation in vitro. GST-tagged NEMO without its N-terminal IKK-binding region (GST-NEMOΔN) was mixed with cytosolic extract from Nemo−/− MEF cells to collect GST-NEMOΔN pull down (NEMOΔN PD). This material, or GST-NEMO, was incubated with cytosolic extract (S100) from HeLa cells depleted of NEMO with a NEMO antibody. Activation of IRF3 was analyzed as described in (B). (D) Reconstitution of IRF3 dimerization in vitro. NEMOΔN PD, His8-E1, Ubc5c, His6-TRAF6, ubiquitin, His6-MAVSΔTM, His8-IRF3, and 35S-Flag-IRF3 were incubated together with ATP as indicated, followed by analysis of IRF3 dimerization. (E and F) TRAF6 is important for IRF3 and NF-κB activation by MAVS in vitro. Cytosolic extracts from wild-type or Traf6−/− MEF cells were incubated with His6-MAVSΔTM together with WT or mutant TRAF6 protein as indicated, followed by analysis of IRF3 dimerization (E) or IκBα phosphorylation (F). T6RZC: TRAF6 containing the RING, zinc, and coiled-coil domains, with the TRAF-C domain replaced by a fragment of bacterial gyrase B. (G) TRAF2 and 5 are important for IRF3 activation by MAVS in vitro. Cytosolic extracts from WT or different TRAF deficient MEF cells were incubated with His6-MAVSΔTM, followed by IRF3 dimerization assay. (H and I) Either TRAF2 or TRAF5 rescues IRF3 and IKK activation by MAVS in the Traf2/5 DKO extract. Traf2/5 DKO extracts were supplemented with TRAF2 or TRAF5 proteins as indicated, together with His6-MAVSΔTM and ATP, followed by measurement of IRF3 dimerization and IκBα phosphorylation.