Identification of Sp1 binding to the MR promoter by electrophoretic mobility shift analysis. A double-stranded MR promoter oligonucleotide (bp −90 to −125) that included the single Sp1 site was labeled with [γ-³²P]-dATP and incubated with either nuclear extract (U) or with purified Sp1 protein (Promega) in the presence of 2 μg of double-stranded poly (dI:dC) under conditions described in the Materials and Methods. Unlabeled double-stranded competitor oligonucleotides of each promoter segment were added at a 50-molar excess over the probe oligonucleotide (lanes 2 and 5). The antibody supershift assay was performed by preincubating the binding protein for 15 minutes with anti-Sp1 antibody (Santa Cruz Biotechnology) before the addition of the probe for an additional 20 minutes. The arrows on the left of the diagram indicate the positions of the Sp1 binding complex (lanes 1 and 4) and the antibody supershifted complex (lane 3).