FIG. 4. .

Novel DNA-binding complexes induced in mutant IFNAR-2c (FF) cells by interferon-β (IFN-β). (A) The nucleotide sequence of the wild-type GATE element (wtGATE), mutated GATE element (mGATE, mutated residues are shown underlined), and the consensus sequence for an ISRE are shown. The nucleotide residues not conserved between ISRE and GATE are indicated by thick vertical lines and thin vertical lines indicate consensus. (B) EMSA analysis of nuclear extracts from FF and wild-type IFNAR-2c (R2C) cells treated with recombinant IFN-β (5,000 U/mL) for 2 or 5 h or reserved as untreated controls (−) were assayed. The ³²P-labeled GATE probe bound 2 complexes, BBF-1 and BBF-2. Results from one of three experiments are shown. Incubation with a 50-fold molar excess of unlabeled wild-type GATE oligonucleotide (wtGATE) competed out the complexes but not mutated GATE oligonucleotide (mGATE) prior to incubation with radiolabeled GATE probe. (C) IFN-β–induced DNA-binding complexes required de novo protein synthesis. FF and R2C cells were pretreated with cycloheximide (100 µg/mL) for 30 min followed by treatment with or without IFN-β (5,000 U/mL) for 6 h or reserved as untreated controls.