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. 2011 Dec 9;287(5):3313–3325. doi: 10.1074/jbc.M111.309021

FIGURE 4.

FIGURE 4.

KD548-Fc induces a DR4/DR5-associated signaling complex composed of RFK, Nox1, Rac1, TRADD, and TRAF2 in HeLa cells, where RFK plays an essential role in recruiting Nox1 to DR4/DR5. A, Western blots (WB) showing immunoprecipitation (IP) of the lysates of TRAIL- and KD548-Fc-treated cells using anti-FLAG antibody (for TRAIL) and anti-human Fc antibody (for KD548-Fc), respectively. Cells were left untreated (control) or treated with FLAG-tagged TRAIL (30 nm) or KD548-Fc (0.8 μm) for 0.5 or 2 h and then subjected to immunoprecipitation and/or Western blot. B, Western blots showing KD548-Fc immunoprecipitation of the lysates of DR4-, DR5-, or DR4/DR5-depleted cells using anti-human Fc antibody after KD548-Fc (0.8 μm) treatment for 0.5 or 2 h or no treatment (control). C, Western blots showing DR4 and DR5 immunoprecipitation of the cell lysates using anti-DR4 and anti-DR5 antibody, respectively, after KD548-Fc (0.8 μm) treatment for 0.5 or 2 h or no treatment (control). D, Western blot analysis of the expression levels of RFK, TRADD, and TRAF2 in wild type, RFK-deficient HeLashRFK, TRADD, and TRAF2 siRNA-transfected HeLa cells, respectively. Control, untransfected cells. E, Western blots showing immunoprecipitation of the lysates of TRAIL- and KD548-Fc-treated RFK-deficient HeLashRFK cells using anti-FLAG antibody (for TRAIL) and anti-human Fc antibody (for KD548-Fc), respectively. Experiments were performed as described in A. F, Western blots showing KD548-Fc immunoprecipitation of the lysates of TRADD- or TRAF2-depleted cells using anti-human Fc antibody after KD548-Fc (0.8 μm) treatment for 0.5 or 2 h or no treatment (control). G, Western blots showing RFK immunoprecipitation of the cell lysates using anti-RFK antibody after KD548-Fc (0.8 μm) treatment for 0.5 or 2 h or no treatment (control). H, superoxide anion generation assay in wild-type and RFK-deficient HeLashRFK cells by KD548-Fc (0.8 μm) treatment. Control, untreated wild type HeLa cells. I, Western blots showing immunoprecipitation of the lysates of Rac1-, Noxo1-, or Noxa1-depleted cells using anti-human Fc antibody after KD548-Fc (0.8 μm) treatment for 2 h or no treatment (control).