Mutational analysis of the −164 and −177 PU.1 sites. Oligonucleotide probes were prepared in which the PU.1 sites at −164 and −177 were mutated (GAGGAA to GACTAA) either separately (P177M, lanes 4 through 6; and P164M, lanes 7 through 9) or in combination (P177/164M, lanes 10 through 12). Binding assays of nuclear extract (U) and in vitro-translated PU.1 (i.v. PU) to these probes were performed as described in the Materials and Methods and compared with binding to the unmutated wild-type oligonucleotide probe (−184/−150, lanes 1 through 3). Unlabeled double-stranded competitor oligonucleotides were added at 50-molar excess over the probe oligo (lanes 2, 5, 8, and 11). The arrow on the left indicates PU.1 binding to the oligonucleotide probes.