Inhibitory effects of celastrol. (A) Celastrol inhibits TNF-induced degradation of IκBα. KBM-5 cells were incubated with 5 μM celastrol for 6 hours 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. The results shown are representative of 2 or 3 independent experiments. Equal protein loading was evaluated by β-actin (bottom). (B) Celastrol blocks the phosphorylation of IκBα by TNF. Cells were preincubated with 5 μM celastrol for 6 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 using phospho-specific anti-IκBα antibody. The same membrane was reblotted with β-actin antibody. (C) The effect of celastrol on the activation of IKK by TNF was investigated. KBM-5 cells were incubated with 5 μM celastrol for 6 hours, incubated with 50 μg/ml ALLN for 30 minutes, and then treated with 1 nM TNF for different time intervals. Whole-cell extracts were prepared, and extracts were immunoprecipitated with antibodies against IKK-α and IKK-β. Thereafter, the immune complex kinase assay was performed as described in “Materials and methods.” To examine the effect of celastrol 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 and anti–IKK-β antibodies. The results shown are representative of 3 independent experiments. (D) Celastrol inhibits TNF-induced nuclear translocation and phosphorylation of p65. KBM-5 cells were either untreated or were pretreated with 5 μM of celastrol for 6 hours at 37°C and then treated with 0.1 nM TNF for the indicated times. Nuclear extracts were prepared and analyzed by Western blotting using antibodies against phospho-specific p65 and anti-p65. The results shown are representative of 2 or 3 independent experiments.