The COX-2-selective NSAID NS-398 induces NF-κB DNA-binding activity in the form of p50/p50 and p50/p65 in HT-29 cells. HT-29 cells were treated with 0 or 50 μ M NS-398 for 96 h. Nuclear extracts were prepared and 4 μg analysed by supershift EMSA in the presence or absence of antibodies to NF-κB proteins p50, p65, p52, c-Rel or RelB or control IgG. The p50 antibody supershifts both bands, whereas the anti-p65 antibody only affects the upper band, suggesting that the lower band consists of p50/p50 and the upper band of p50/p65. The p65 antibody does not produce a supershifted band, presumably due to interference with DNA binding. This experiment has also been carried out using samples treated with 60 μ M NS-398 for 72 h with equivalent results.