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MAVS activates TBK1 and IKKε through TRAFs in NEMO dependent and independent manner

Fig 3

MAVS activates TBK1/IKKε in both NEMO-dependent and independent manner.

(A) WT and NEMO−/− 293T cells were infected with SeV for the indicated times. Cell lysates were immunoprecipitated with the anti-MAVS antibody. The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies. (B) and (C) WT, MAVS−/− and NEMO−/− 293T cells 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 Quantitative–PCR analysis of Ifnβ induction (C). (D) WT and NEMO−/− HeLa cells were infected with SeV for the indicated times. Type I-IFN and IL-6 production was determined by bioassay or ELISA respectively. (E) and (F) WT and Nemo−/− MEFs were infected with SeV for the indicated times. Cells were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (E), or Quantitative–PCR analysis of Ifnβ induction (F). (G) WT, MAVS−/− and NEMO−/− 293T cells were transfected with luciferase reporter constructs (IFNβ–Luc or IFNβ (dNF-κB)-Luc or p561-Luc, 50ng) and the indicated plasmids (empty vector 100 ng, RIG-I-N 100 ng, MAVS 50 ng and TBK1 50 ng). Luciferase assay was performed after 24 h. (H) WT, MAVS−/− and NEMO−/− 293T cells were transfected with IFNβ–Luc reporter (50 ng) and the indicated plasmids (RIG-I-N 100 ng, empty vector 200ng, NEMO 200 ng, IKKβ 200 ng, IKKα+β 100 ng each). Luciferase assay was performed after 24 h. Data from (C), (D), (F), (G) and (H) represent mean ± SD. Similar results were obtained in 3 independent experiments. See also S4 Fig.

Fig 3

doi: https://doi.org/10.1371/journal.ppat.1006720.g003