Mavs −/− MEF cells stably expressing Flag-tagged wild-type MAVS or its mutants were mock-treated or infected with SeV for 12 hr. (A) As in Figure 5—figure supplement 1, MAVS puncta formation and redistribution were examined using confocal microscopy. Mitochondria were stained by TOM20 antibody (mitochondrial outer membrane). Areas within the yellow windows in the merged images are expanded and shown in the rightmost images. MAVS puncta are highlighted by white arrowheads. Scale bars, 5.0 μm. (B) Statistical analysis of the correlation between nuclear translocation of p65 and SeV-induced MAVS puncta formation in Mavs−/− MEF cells expressing wild-type MAVS. No p65 nuclear translocation was seen in uninfected cells. Among cells showing clear p65 translocation, only those showing large MAVS puncta (probably > 0.4 μm) were classified as positive. Even with such strict constraints, more than 70% of the cells were positive. (C) IFNβ production was rescued by expressing MAVS wild-type and mutants (E80A and F16H) that retain the capability of forming MAVS puncta, but not those (E26A, F16A and Y30A) that failed to form puncta (Figure 5—figure supplement 1). The IFNβ mRNA level was quantified by q-RT-PCR. (D) MAVS proteins in the mitochondrial extracts from different cells were separated by SDD-AGE (top) or SDS-PAGE (bottom) followed by western blotting. Cells expressing wild-type MAVS and mutants (E80A, L48A, F16H, W56Y, Y30F and R77A) that rescued IFNβ production gave rise to strong aggregate signal in the SDD-AGE assay, whereas those expressing MAVS mutants defective in forming puncta or inducing IFNβ (E26A, R64A, R65A, F16A or Y30A) did not form aggregates. Y30A showed slightly higher basal aggregation signal, independent of viral infection.