FIGURE 4.

(A) Sesamin inhibits TNF-induced activation of NF-κB. KBM-5 cells were incubated with 100 μM sesamin for 12 hr, treated with 0.1 nM TNF for the indicated time intervals, and then analyzed by EMSA for NF-κB activation. (B) Effect of sesamin on TNF-induced degradation of IκBα. KBM-5 cells were incubated with 100 μM sesamin for 12 hr and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared and analyzed by Western blotting using antibodies against anti-IκBα. Equal protein loading was evaluated by β-actin. (C) Effect of sesamin on phosphorylation of IκBα induced by TNF. Cells were preincubated with 100 μM sesamin for 12 hr, incubated with 50μg/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 30 min, and then treated with 0.1 nM TNF for 10 min. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using phospho-specific IκBα antibody. (D) Effect of sesamin on the activation of IKK by TNF. (Left) KBM-5 cells were preincubated with 100 μM sesamin for 12 hr, and then treated with 1 nM TNF for the indicated time points. Whole cell extracts were immunoprecipitated with antibody against IKK-α and analyzed by an immune complex kinase assay. To examine the effect of sesamin on the level of expression of IKK proteins, whole cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α antibody. (Right) Direct effect of sesamin on IKK activation induced by TNF. Whole cell extracts were prepared from KBM-5 cells treated with 1 nM TNF and immunoprecipitated with anti-IKK-α antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentration of sesamin.