PU.1 interacts with a PU box in the c-myb promoter. (A) Wild-type and mutated sequences of the PU box–containing oligonucleotides corresponding to nucleotides +6 to +40 of the human c-myb promoter. The mutated nucleotides are indicated by asterisks. (B) EMSA of PU.1 binding to the c-myb promoter. The double-stranded c-myb wild-type oligonucleotide was ³²P-labeled and incubated with GST protein (lane 2), GST-PU.1 recombinant fusion protein (lanes 3 and 6), GST-PU.1 in the presence of a 100-fold molar excess of wild-type (lane 4) or mutated (lane 5) unlabeled probe, and GST-PU.1 in the presence of the anti-PU.1 antibody (lane 7). (B) EMSA were also performed with GST or GST-PU.1 and the PU box–mutated c-myb promoter fragment as probe (lanes 8 and 9). (C) EMSA performed with whole cell extracts from G-CSF–treated 32Dcl3 cells and PU box–containing c-myb double-stranded oligonucleotide as probe. Binding was performed in the presence of an anti-PU.1 antibody (lanes 5 to 8) or in the presence of a nonrelated rabbit polyclonal antibody (lanes 1 to 5). Binding was also performed in the presence of a 100-fold molar excess of either unlabeled wild-type or mutated PU box–containing c-myb double-stranded oligonucleotide used as specific or nonspecific competitor (lanes 15 and 16), respectively. (D) EMSA with whole cell extracts from U937 and Jurkat cells were performed as controls (lanes 1 to 4). PU.1 indicates the DNA/PU.1 protein complex; PU.1(ss) indicates the supershifted DNA-PU.1/anti-PU.1 Ab complex.