Identification of PU.1 binding to the MR promoter by gel mobility shift analysis. Oligonucleotide probes representing promoter segments that included each of the four potential PU.1 sites were prepared as described in the Materials and Methods. Binding reactions were performed on ice for 30 minutes by incubating the respective probes with either 5 μg of nuclear extract (U) or 1 μL of in vitro-translated PU.1 protein (i.v. PU) in the presence of 2 μg of double-stranded poly (dI:dC). The source of nuclear extract in the assay portrayed was from U937 cells. The probe −212/−185 contains the ETS-1 site at −204 (lanes 1 through 3); the probe −184/−150 contains the pair of PU.1 sites at −164 and −177 (lanes 4 through 6); the probe −39/−15 contains the pair of PU.1 sites at −32 and −22 (lanes 7 through 9); similarly, the probe −39/+10 also contained the −32 and −22 PU.1 sites but in addition included the initiator element (Inr) overlapping the transcription start site (lanes 10 through 12; see Fig 1). Unlabeled double-stranded competitor oligonucleotides of each promoter segment were added at 50-molar excess over the probe oligo (lanes 3, 6, 9, and 12). The arrow on the left indicates the complex of PU.1 binding to the −184/−150 oligonucleotide.