GG->CC mutation in the CEACAM1 ISRE impairs IRF-1 and IRF-2 Binding. (A) CEACAM1 ISRE (underline) containing 60-mer oligonucleotides are shown with enlarged nt (GG) in the WT or (CC) in the mutant. EBNA DNA served as the positive control for binding. (B-D) Fluorescent electrophoretic mobility shifts (fEMSA) were performed with 0.3 pmol IRDye-labeled oligonucleotides (EBNA-700 nm, ISRE GG->CC-800 nm and ISRE WT-700 nm) and increasing moles of IRF-1 (B) or IRF-2 (C) as shown. Epstein-Barr Nuclear Antigen extract is used to form a positive control complex denoted by EBNA complex (left). DNA-protein complexes produced by incubation with specific IRF proteins produce either a slow-migrating (C1) or faster-migrating complex (C2). (D) Competition assays were performed by adding increasing amounts of ISRE-WT DNA (red channel) in the presence of 0.3 pmol ISRE GG->CC DNA (green channel) and either 67 pmol IRF-1 or IRF-2 protein. Addition of competitor DNA to produce all three binding states (unbound, yellow C2 and C1 complexes) indicates complete saturation of exogenous IRF protein. Results are from one of three experiments with similar results.