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

Fig 2

TBK1/IKKε are recruited to MAVS via the pre-associated TRAFs-TBK1/IKKe.

(A) 293T cells deficiency of TRAF2, 3, 5, or 6 or TRAFs-deficient cells reconstituted with TRAF6 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) The reconstituted cells described in Fig 1G were infected with SeV for the indicated times. Cell lysates were immunoprecipitated with the anti-HA antibody. The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies. (C) The reconstituted cells described in Fig 1B were infected with SeV for the indicated times. Cell lysates were immunoprecipitated with the anti-Flag antibody. The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies. (D) Cell lysates of 293T, HeLa, and THP1 cells were immunoprecipitated with the anti-TRAF2, 3, or 6 antibodies. The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies. (E) Recombinant TBK1-Flag and His-tagged TRAFs (TRAF2C: containing amino acids 349–496) were pulled down with Ni-NTA beads. The precipitates and input were analyzed by Western blot with the indicated antibodies. (F) 293T cells were transfected with Flag-tagged TRAFs and full length TBK1 or TBK1 truncations illustrated in the upper panel for 24 h. Cell lysates were immunoprecipitated with the anti-Flag antibody. The precipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies. Truncations 1 to 4 indicate full length TBK1 and TBK1 lacking amino acids 309–385, 618–657 and 658–745. See also S3 Fig.

Fig 2

doi: https://doi.org/10.1371/journal.ppat.1006720.g002