Physical and functional characterization of 2 Sp1-binding sites within the IL-12(p35) promoter. (A) Competition assays. Nuclear extracts (10 μg protein) were prepared from DCs and incubated with [³²P]-radiolabeled Sp1#1wt and Sp1#2wt probes in the absence of competitor or in the presence of increasing concentrations (5- to 80-fold molar excess) of the indicated unlabeled competitors. The figure shows only the specific retarded complex. (B) The major retarded complex is predominantly composed of Sp1. Supershift assays were performed by incubating Sp1#1wt and Sp1#2wt probes with DC nuclear extracts in the absence of antibody, or in the presence of anti-Sp1, anti-Sp3, or as a control, anti–MEF-2 antibodies. (C) Effect of the mutations in the Sp1 sites on IL-12(p35) gene expression. Unstimulated and IFN-γ/LPS-stimulated RAW 264.7 cells were transiently transfected with 2 μg p35-lucWT, p35-mutSp1#1, or p35-mutSp1#2 plasmids and 20 ng pRL-TK as an internal control plasmid. Promoter activities were normalized using Renilla luciferase activities. Values represent the means ± SD of triplicate samples performed in the same experiment. A representative experiment of 3 independent experiments is shown. RLU indicates relative light units. (D) Ectopic expression of Sp1 transcription factor up-regulates p35 promoter activity. SL2 cells were transiently cotransfected with p35-lucWT (1 μg) and increasing amounts of pPacSp1. To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of plasmid DNA were complemented to 1.6 μg total DNA by using the empty pPac plasmid. Results are presented as histograms indicating the induction by Sp1 (in fold) with respect to the activity of the p35-lucWT in the absence of Sp1, which was assigned a value of 1. Values represent the means ± SD of triplicate transfection performed in the same experiment. A representative experiment of 4 independent experiments is shown.