Figure 2

Activation of NF-κB DNA-binding activity in colorectal carcinoma cells by the COX-2-selective NSAID NS-398. (A) NS-398 induces NF-κB DNA-binding activity in HT-29 cells. HT-29 cells were treated with 0, 50 or 75 μ M NS-398 for 72 or 96 h. At the indicated time points, nuclear extracts were prepared and 1 μg analysed by EMSA. Competition assay (right-hand panel): +NF-κB=plus unlabelled NF-κB oligonucleotide (specific competitor), +AP-1=plus unlabelled AP-1 oligonucleotide (nonspecific competitor), −=no oligonucleotide. The final lane contains no nuclear protein extract as a negative control. (B) NS-398 downregulates IκBα levels in HT-29 cells. HT-29 cells were treated with 0, 50 or 75 μ M NS-398 for 72 or 96 h. Whole-cell lysates from 10⁶ attached cells were analysed by Western blotting for IκBα expression levels at 72 and 96 h. Blots were probed with an α-tubulin antibody as a control for equal loading and transfer. Results shown are representative of those obtained in three independent repeat experiments.