AZT, but not GCV, inhibits the binding of nuclear proteins to oligonucleotides containing NF-κB sites. (A) Purified nuclear extracts (10 μg) prepared from EBV⁺ BL cell lines (BL-8, BL-5) and EBV^- BL cell lines (BL-9, BL-10) were preincubated with the indicated antibodies to the NF-κB components before the addition of radiolabeled probes. The composition of the NF-κB complex in EBV⁺ and EBV^- BL cells was characterized by supershift analysis. Supershift indicates the binding of antibody and protein-DNA complex. (B) Nuclear extracts prepared from AZT (10 μg/mL), GCV (10 μg/mL), Bay 11-7082 (2.5 μM), or sodium butyrate (2.5 mM) treated cells (BL-5, BL-8, BL-9) for the indicated times were assayed by EMSA using either NF-κB (top blot) or Oct-1 (bottom blot) specific consensus oligonucleotides as probes. The results are representative of 3 independent experiments. Oct-1 was used as a loading control for nuclear proteins. (C) BL-5 and BL-8 cells were treated with AZT (10 μg/mL) for the indicated times and p50 was detected in nuclear extracts by ELISA. Specificity of binding was confirmed by using both wild-type and mutated NF-κB oligonucleotides. Experiments were performed in triplicate. Results are shown as the mean ± standard deviation.