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. 2012 Sep 24;209(10):1703–1711. doi: 10.1084/jem.20120024

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© 2012 Zhang et al.

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Figure 3. — TRIP Targets TBK1 for degradation. (A) HEK293 cells were transfected with RIG-I, MAVS, TBK1, TRIF, and MDA5 along with TRIP plasmid, and IFN-β mRNA was analyzed by RT-PCR. (B) HEK293 cells were transfected with TRIF, RIG-I, MAVS, TBK1, and IRF3 5D along with IFN-β reporter plasmid and TRIP plasmid, and luciferase activity was analyzed. (C) Western blot analysis of the expression of TBK1, IRF3, STAT1, and TRAF3 in peritoneal macrophages transfected with control siRNA or TRIP siRNA and then stimulated with LPS or poly (I:C) for 6 h. (D) Western blot analysis of the expression of TBK1, TRAF3, and TRAF6 in RAW264.7 cells stably transfected with control or HA-TRIP plasmid and then stimulated with LPS for indicated time periods. (E) Western blot analysis of TBK1 or TRAF3 expression in HEK293 cells cotransfected with HA-TBK1 or Myc-TRAF3 and Flag-TRIP or vector control and then treated with proteasome inhibitor MG-132 for 4 h. (F) In vitro kinase assays of TBK1 in the lysates of peritoneal macrophages transfected with control siRNA or TRIP siRNA 1 and then stimulated with LPS for 30 min, assessed with the substrates MBP (for TBK1). **, P < 0.01. Data are representative of three experiments (mean and SD of six samples in B and F). Similar results were obtained in at least three independent experiments in A, C, D, and E.