MAVS activates TBK1 and IKKε through TRAFs in NEMO dependent and independent manner
(A) WT and TRAF2−/−, TRAF3−/−, TRAF5−/−, TRAF6−/− 293T cells were infected with SeV for the indicated times. Type I-IFN production was determined by bioassay. For each knockout cell, two independent clones were accessed with similar results. (B) to (E) WT 293T cells, 293T cells deficiency of TRAF2, 3, 5, or 6 or cells deficiency of TRAF2, 3, 5, and 6 (TRAFs-deficient) reconstituted with TRAF2, 3, 5, or 6 individually were infected with SeV for the indicated times. Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (B), or RT-PCR analysis of the indicated gene induction (C), bioassay analysis of type I-IFN production (D) and ELISA analysis of IL6 production (E). (F) WT and NEMO−/− 293T cells were transfected with IFNβ–Luc reporter (50 ng) and the indicated plasmids (RIG-I-N 100 ng, MAVS 50 ng, or TBK1 50 ng). Luciferase assay was performed after 24 h. (G) to (H) WT, MAVS−/− THP1 cells and MAVS−/− THP1 cells reconstituted with MAVS-WT, MAVS-d2 (Q145N), MAVS-d6 (E155D, E457D), or MAVS-d2&6(Q145N, E155D, E457D) were infected with SeV for the indicated times. Type I-IFN production was determined by bioassay (G). Phosphorylation and expression of the indicated proteins were detected by Western blot (H). Data from (A), (D), (E), (F) and (G) represent mean ± SD. Similar results were obtained in 3 independent experiments. See also S2 Fig.