Specificity tests for NF-κB binding. EMSA was performed using extracts from macrophages stimulated with TNF for 2 h. Unlabeled probe in excess (as indicated below) was used as specific competitor. The mutant competitor (see Methods), had one point mutation in the NF-κB binding site. The antibodies (2 μg/sample) were added after the radioactive probe. After addition of antibodies, incubation was continued for 1 h on ice. Lanes: 1, probe only; 2, control (TNF, 10 ng/mL, 2 h); 3, specific competitor (50X excess); 4, specific competitor (100X excess); 5, mutant competitor (50X excess); 6, mutant competitor (100X excess); 7, anti-p65 (RelA); 8, anti-p50 (NFKB1). The arrowheads indicate the positions of specific complexes. The bars mark supershifted bands. Data are representative of four separate experiments.