Fig 3.

TRAF2 Ser11 phosphorylation recruits the IKK complex and RIP1. (A) TRAF2 interacts with the canonical IKK complex in HEK293T cells. HEK293T cells were cotransfected as indicated with either wild-type Myc-TRAF2 or Myc-TRAF2-S11A and V5-tagged IKKα, IKKβ, and IKKγ (input, right). Myc-TRAF2 immune complexes were isolated and analyzed with antibodies specific for TRAF2 and V5 (left). (B) Endogenous interaction between of TRAF2, IKKβ, IKKγ, RIP1, and cIAP1. TRAF2 immune complexes were isolated from MCF-7 cells and analyzed with the indicated antibodies following treatment with TNF-α (20 ng/ml for 30 min). (C) TRAF2 interactions with the IKK complex, cIAP1, and RIP1 are dependent on phosphorylation. MCF-7 cells were transduced with V5-TRAF2 or V5-TRAF2 S11A. V5-TRAF2 immune complexes were isolated and analyzed by immunoblotting for endogenous IKKβ, IKKγ, RIP1, and cIAP1.