The p40^phox PU.1 sites bind PU.1 with different avidity.

(A) EMSA of the p40^phox PU.1 sites with varied amounts of the DNA probes. Increasing amounts of the³²P-labeled PU.1 DNA probes having comparable specific activities of labeling were incubated with in vitro–synthesized PU.1 protein and then analyzed by PAGE. The major PU.1-DNA complex is indicated by the arrow. (B) Competition between oligonucleotides corresponding to the p40^phox PU.1 binding sites and a ³²P-labeled PU.1 DNA probe (5′-CAAAAGCGACTTCCTCTTTCCAGTGC-3′) from thep47^phox promoter for binding to PU.1 protein in HL-60 nuclear extracts. Fixed amounts of HL-60 nuclear extract and ³²P-labeledp47^phox PU.1 probe were incubated in the presence of the indicated amounts of the unlabeledp40^phoxoligonucleotides. Specificity of the PU.1-DNA complex indicated by the arrow was confirmed by supershift assay with antibodies to human PU.1 (data not shown).