Figure 2

IKK activity was not obviously inhibited by H₂O₂. (A) HEK293 cells were exposed to various doses of H₂O₂ for 20 min, washed and stimulated with TNFα (20 ng/ml) for 10 min. IKK complexes in the cell lysate were immunoprecipitated with anti-IKKγ antibody. An in vitro kinase assay (KA) was carried out using [γ-³²P]ATP and GST-IκBα(1-54) as substrates. The IKKγ in the kinase assay mixture was measured by immunoblot (IB) analysis. (B) The cells were exposed to H₂O₂ (0.5 mM) for 20 min and stimulated with TNFα for various times. IKK activity was determined as in (A). (C) The cells were exposed to H₂O₂ (0.5 mM) for 20 min and stimulated with TNFα for 10 min. The cells were lysed in cell lysis buffer containing various concentrations of DTT. Immunoprecipitation and in vitro kinase assays were performed as in (A) using buffers containing the indicated concentrations of DTT.