Celastrol blocks NF-κB activation induced by TNF in a dose- and time-dependent manner. (A) KBM-5 cells were incubated with the indicated concentrations of celastrol for 6 hours and then treated with 0.1 nM TNF for 30 minutes. The nuclear extracts were assayed for NF-κB activation by EMSA. The results shown are representative of 3 independent experiments. (B) KBM-5 cells were preincubated with 5 μM celastrol for the indicated times and then treated with 0.1 nM TNF for 30 minutes. The nuclear extracts were prepared and assayed for NF-κB activation by EMSA. The results shown are representative of 3 independent experiments. (C) NF-κB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated cells or cells treated with 0.1 nM TNF were incubated with the indicated antibodies, an unlabeled NF-κB oligoprobe, or a mutant oligoprobe. They were then assayed for NF-κB activation by EMSA.