FIG. 2.
IRF-3a does not bind to the ISG15 ISRE or the PRD I/III element. (A) Comparable amounts (5 μl) of in vitro-translated IRF-3, Xpress-IRF-3 (Xp-IRF-3), IRF-3a, and AU1-IRF-3a were used for EMSA along with a 32P-labeled probe corresponding to the ISRE of the ISG15 gene (5′-CTCGGGAAAGGGAAACCGAAACTGAAGCC-3′). Ab, antibody. Xpress and AU1 are epitope tags. (B) Fifty nanograms (lanes 2 and 8 to 11) or 5 ng (lanes 3 to 7) of full-length GST–IRF-3 or GST–IRF-3a was used for EMSA with a 32P-labeled probe corresponding to the PRD I-PRD III region of the IFN-β promoter (5′-GAAAACTGAAAGGGAGAAGTGAAAGTG-3′). In all cases, an anti-IRF-3 antibody (SL-12) or a nonspecific control antibody was added to the binding reaction to demonstrate the specificity of the complexes. Cold probe was added at a 100-fold excess. Note that the difference in complex migration between the two gels reflects the difference in the type of gel used.