Partial inhibition of NS-398-induced NF-κB DNA-binding activity does not enhance apoptosis induction in HT-29 cells. HT-29 cells were infected with 10 MOI (for NS-398 experiments; left-hand panel) or 50 MOI (for TNF-α experiments; right-hand panel) of either the control vector rAd.βgal or rAd.IκBα and treated the following day with 60 μ M NS-398 for 72 h or 100 ng ml−1 TNF-α for 24 h (as TNF-α is a more rapid inducer of apoptosis). (A): Increased expression of IκBα in HT-29 cells infected with rAdIκBα. Whole cell lysates from 106 attached cells were prepared and analysed by Western blotting for IκBα expression levels. Blots were probed with an α-tubulin antibody as a control for equal loading and transfer. (B): Inhibition of NF-κB DNA binding by rAdIκBα. Nuclear protein extracts were prepared and 1 μg analysed by EMSA. (C): Infection with rAd.IκBα potentiates apoptosis induced by TNF-α but not NS-398. Both attached and floating cells were harvested and counted. Floating cell yields were calculated as a percentage of total cell yields and are shown as mean±s.d. Results shown in panels A–C for NS-398 and for TNF-α are from within the same experiment and are representative of two independent experiments carried out in duplicate. 1=rAd.βgal; 2=rAd.βgal+NS-398; 3=rAd.IκBα; 4=rAd.IκBα +NS-398; 5=rAd.βgal; 6=rAd.βgal+TNF-α; 7=rAd.IκBα; 8=rAd.IκBα +TNF-α.