Physical characterization of the ISRE-1 site within the IL-12p35 proximal promoter region. (A) IRF-3 consensus DNA-binding sequence is shown.21 The oligonucleotide used as probe in EMSA (named ISREwt) encompasses 2 overlapping putative ISRE sites (ISRE-1 and -2, indicated by the underlined nucleotides). An asterisk indicates nucleotides that differ from the consensus. For ISREMut A, B, and C probes, only the bases that are changed compared with ISREwt are indicated. (B) Competition assays. Recombinant N-terminal IRF-3 (2 ng) was incubated with radiolabeled ISREwt probe in the absence or presence of 12.5- and 50-fold excess of the indicated unlabeled competitor. (C) EMSA was performed with decreasing amounts of IRF-3 protein (10, 2, and 0.4 ng) and radiolabeled ISREwt, ISRE Mut A, Mut B, and Mut C probes.
