Characterization of 3 PU.1 binding sites in the p40^phox promoter.

(A) Sequence of the proximal promoter region of thep40^phox gene. 3 PU.1 sites are underlined and labeled PU.1a, PU.1b, and PU.1c. The arrow indicates the reported transcription start site,36 and the translation initiation codon is double-underlined. Nested reverse primers used in cloning are in bold font. (B) Sequences of wild-type and mutatedp40^phox PU.1 oligonucleotides used in the EMSA studies. Core PU.1 binding domains are underlined, and mutated nucleotides are shown in bold. (C) EMSA of HL-60 nuclear extracts with³²P-labeled p40^phox PU.1 DNA probes. HL-60 nuclear extract (5 μg) was incubated with the labeled probes alone (lanes 1, 5, and 9) or together with either antibody to PU.1 (lanes 2, 6, and 10) or a 200-fold molar excess of the homologous wild-type (lanes 3, 7, and 11) or mutated (lanes 4, 8, and 12) oligonucleotides (Figure 2B). The specific PU.1-DNA complex (PU.1) and the supershifted complex (SS) are indicated by arrows.