FIGURE 2.

Exposure to H₂O₂ diminishes SCF^β-TrCP-dependent ubiquitination of IκBα. A and B, levels of ubiquitinated IκBα (Ub_n-IκBα) were determined in neutrophils (A) or in 293-hTLR4/MD2-CD14 cells that transiently expressed Myc-tagged IκBα, IKKβ-FLAG, or Ub-HA. In B, cells were treated with combinations of H₂O₂, LPS, and MG132 as indicated, and cell extracts were subjected to immunoprecipitation (IP) with anti-ubiquitin antibodies. Representative Western blots show the amounts of Ub_n-IκBα conjugates detected with antibodies specific for phospho-IκBα. Two additional experiments provided similar results. C and D, neutrophils (C) or 293-hTLR4/MD2-CD14 cells stably expressing β-TrCP-FLAG (D) were cultured with H₂O₂ (0 or 300 μm) for 60 min, and then the SCF^β-TrCP complex was immunoprecipitated from cell extracts using anti-cullin-1 antibodies, followed by determination of SCF^β-TrCP-dependent ubiquitination of phosphorylated recombinant IκBα. Representative Western blots show levels of Ub_n-IκBα as detected with anti-phospho-IκBα antibodies. A second experiment provided similar results.