Celastrol suppresses TNF-induced NF-κB activation in different cell types. (A) Human lung carcinoma H1299 and (B) human embryonic kidney A293 cells were incubated with 5 μM celastrol for 6 hours, followed by incubation with 0.1 nM TNF for 30 minutes. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. The results shown are representative of 3 independent experiments. (C) Human multiple myeloma U266 and (D) bladder cancer 253JBV cells were incubated with the indicated concentrations of celastrol for 6 hours. Nuclear extracts were then prepared and analyzed for NF-κB activation by EMSA. (E) The direct effect of celastrol on NF-κB complex was investigated. Nuclear extracts were prepared from untreated cells or cells treated with 0.1 nM TNF and incubated for 30 minutes with the indicated concentrations of celastrol. They were then assayed for NF-κB activation by EMSA. The results shown are representative of 3 independent experiments.