E₂ decreases AP-1 binding to the TRE site in the TNF promoter, whereas it does not affect the binding of NF-κB and Sp-1 to their consensus sequences. EMSAs were performed by incubating nuclear extracts of untreated and E₂-treated RAW 264.7 cells stimulated with IL-1 and TNF for 1 hour with ³²P-labeled probes containing the TRE, the NF-κB, or the Sp-1 binding sites in the TNF promoter. Gel shift complexes were resolved by electrophoresis and viewed by autoradiography. Specificity of the induced complexes was determined by competition with a 50-fold M excess of unlabeled specific probe. Lane 1: cells treated with control vehicle; lane 2: cells treated with E₂ (10^–8 M); lane 3: 50× cold probe; lane 4: free probe. Representative data from 1 of 3 replicate experiments. Densitometric analysis of the data from all experiments revealed that AP-1 binding to the TRE site was approximately 3-fold lower in E₂-treated samples than in vehicle-treated samples.