FIGURE 5.

A20 and TAX1BP1 disrupt a TRAF3-TBK1-IKKi signaling module. A, 293T cells were transfected with either control scrambled siRNA, TRAF3 siRNA, or TRAF6 siRNA. After 24 h, cells were transfected with plasmids as indicated (1 μg of FLAG-IKKi, 500 ng of HA-Ub Lys⁶³-only). Cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. B, TRAF3-deficient MEFs were transfected with either empty vector DNA or a plasmid expressing TRAF3 (2.5 μg). After 36 h, MEFs were mock-infected or virally infected for 16 h. Lysates were subjected to immunoprecipitation and/or immunoblotting with the indicated antibodies. C and D, A20 and TAX1BP1 disrupt TRAF3-IKKi interactions. C, 293T cells were transfected with the indicated plasmids (1 μg of GFP-IKKi, HA-TRAF3, FLAG-A20 WT, FLAG-A20 C103A, or FLAG-A20 1–367). Whole cell lysates were subjected to co-IPs and/or immunoblotting with the indicated antibodies. D, 293T cells were cotransfected with HA-TRAF3 and FLAG-IKKi (1 μg each) and either 1 or 2 μg of GFP-TAX1BP1 plasmid DNAs as indicated. Lysates were subjected to co-IPs and immunoblotting as in C. E and F, 293T cells were transfected with IFNβ luc (100 ng) and pRL-tk (10 ng) DNA together with the indicated plasmids (1 μg each). WT, wild type; IB, immunoblot; IP, immunoprecipitation. Error bars, S.E.