Specificity tests for AP-1 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 2 point mutations in the AP-1 binding site, respectively. 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 (20X excess); 4, specific competitor (40X excess); 5, mutant competitor (20X excess); 6, mutant competitor (40X excess); 7, anti-c-Fos; 8, anti-c-Jun (rabbit); 9, anti-c-Jun (goat); 10, anti-JunB; 11, anti-JunD. The arrowheads indicate the positions of specific complexes. The bars mark supershifted bands. Data are representative of four separate experiments.