FIGURE 2.

3-FC inhibits TNF-α-induced IκBα degradation, IκBα phosphorylation, IKK activation, and p65 nuclear translocation. A, KBM-5 cells were incubated with 3-FC (100 μm) for 12 h and then treated with TNF-α (0.1 nm) for the indicated times. Nuclear extracts were analyzed for NF-κB activation by EMSA. B, effect of 3-FC on TNF-α-induced IκBα degradation. Cells were incubated with 100 μm 3-FC for 12 h and then treated with 0.1 nm TNF-α for the indicated times. Cytoplasmic extracts (CE) were prepared, fractionated on SDS-polyacrylamide gel, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed using anti-IκBα antibody. Anti-β-actin antibody was used as the loading control. C, 3-FC inhibits TNF-α-induced phosphorylation of IκBα. KBM-5 cells were treated with 100 μm 3-FC for 12 h, incubated with 50 μg/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 30 min, and then treated with 0.1 nm TNF-α for 10 min. Cytoplasmic extracts were analyzed by Western blotting using a phospho-specific IκBα antibody (Ser³²/Ser³⁶). The same membrane was reprobed with anti-β-actin antibody. D, 3-FC inhibits TNF-α-induced nuclear translocation and phosphorylation of p65. The nuclear extracts (NE) obtained from cells treated with TNF-α and with or without 3-FC were analyzed by Western blotting using the indicated antibodies (left panel). KBM-5 cells were first treated with 3-FC for 12 h and then exposed to TNF-α (0.1 nm) for 15 min. Cells were then analyzed for p65 localization (right panel). E, effect of 3-FC on TNF-α-induced IKK activation. KBM-5 cells were incubated with 100 μm 3-FC for 12 h and then treated with 1 nm TNF-α for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IKKβ and analyzed using an immune complex kinase assay. The effect of 3-FC on IKK protein expression was determined by Western blot analysis using anti-IKKα and anti-IKKβ antibodies. F, direct effect of 3-FC on TNF-α-induced IKK activation. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nm TNF-α and immunoprecipitated with anti-IKKβ antibody. The immunoprecipitated complex was incubated with the indicated concentrations of 3-FC, and an immune complex kinase assay was performed. G, DTT reverses the inhibitory effect of 3-FC on TNF-α-induced IKK activation. Assays were performed as described from F, except that IKK activity was also determined in the presence of DTT. H, the kinase activity of mutant IKK (C179A) is unaffected by 3-FC. A293 cells were transfected with wild-type FLAG-IKKβ (IKKβ wt) or mutant FLAG-IKKβ (IKKβ mt (C179A)). Whole-cell extracts were prepared, immunoprecipitated, incubated with 3-FC, and subjected to an IKK assay. I, effect of 3-FC on TNF-α-induced TAK1 activation. KBM-5 cells were preincubated with 3-FC for 12 h and then treated with TNF-α (1 nm) for the indicated times. The protein extracts were immunoprecipitated with an antibody against TAK1 and analyzed by an immune complex kinase assay. The results shown are representative of three independent experiments.