GA inhibits NF-κB activation induced by different stimuli. (A) GA blocks NF-κB activation induced by TNF, okadaic acid (OA), PMA, LPS, CSC, and H₂O₂. Human myeloid leukemia KBM-5 cells were preincubated with 5 μM GA for 4 hours and then treated with 0.1 nM TNF for 30 minutes, 500 nM OA for 4 hours, 25 ng/mL PMA for 2 hours, 10 μg/mL LPS, and 40 μg/mL CSC and 250 μM H₂O₂ for 1 hour each. Nuclear extracts were analyzed for NF-κB activation by EMSA. The results shown are representative of 3 independent experiments. (B) Dose-dependent effect of GA on TNF-induced NF-κB activation. KBM-5 cells were incubated with the indicated concentrations of GA for 4 hours and treated with 0.1 nM TNF for 30 minutes. The nuclear extracts were assayed for NF-κB activation by EMSA. CV (%) indicates cell viability of the cells. The results shown are representative of 3 independent experiments. (C) Time-dependent effect of GA on TNF-induced NF-κB activation. KBM-5 cells were preincubated with 5 μM GA 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. CV (%) indicates cell viability of the cells. The results shown are representative of 3 independent experiments. (D) 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. (E) The direct effect of GA on the NF-κB–DNA binding. 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 GA. They were then assayed for NF-κB activation by EMSA. (F) Effect of plumbagin (PG) and GA on the binding of NF-κB to DNA. Nuclear extracts were prepared from untreated cells or cells treated with 0.1 nM TNF and incubated for 30 minutes with the 5-μM concentrations of GA and PG. They were then assayed for NF-κB activation by EMSA.