XN down-regulates the TNF-induced NF-κB activation through inhibition of IκBα kinase. (A) XN inhibits TNF-induced activation of NF-κB. KBM-5 cells were incubated with 50 μM XN for 4 hours, treated with 0.1 nM TNF for the indicated times, and then analyzed for NF-κB activation by EMSA. The fold activation of NF-κB is shown at the bottom. (B) Effect of XN on TNF-induced degradation of IκBα. KBM-5 cells were incubated with 50 μM XN for 4 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared and analyzed by Western blotting using antibody against IκBα. Equal protein loading was evaluated using β-actin. (C) Effect of XN on phosphorylation of IκBα induced by TNF. KBM-5 cells were preincubated with 50 μM XN for 4 hours, incubated with 50 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 30 minutes, and then treated with 0.1 nM TNF for 10 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis with a phosphospecific anti-IκBα antibody (P-IκBα). Ser32/36. (D) Effect of XN on activation of IKK by TNF. KBM-5 cells were preincubated with 50 μM XN for 4 hours and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IKK-α and analyzed using an immune complex kinase assay. To determine the effect of XN on the level of expression of IKK proteins, whole-cell extracts were fractionated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and examined by Western blot analysis with anti–IKK-α and anti–IKK-β antibodies. (E) Direct effect of XN on IKK activation induced by TNF. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nM TNF and immunoprecipitated with an anti–IKK-α antibody. The immunocomplex kinase assay was performed in the absence or presence of XN at the indicated concentrations. (F) Reversal of XN-induced suppression of TNF-induced IKK activation by the reducing agent DTT. Assays were performed as indicated in Figure 4E, except that the IKK activity was determined after treatment with DTT (100 μM), XN (50 μM), or both in kinase assay buffer. (G) The kinase activity of mutated IKK is unaffected by XN. For this, A293 cells were transfected with wild-type FLAG-IKK-β (IKK-β WT) or mutated FLAG-IKK-β (IKK-β MT [C179A]). Whole-cell extracts were prepared, and XN at the indicated concentrations was added in vitro. Immunocomplexes were analyzed for IKK activity.