PU.1 is present in nuclear extracts from HL-60 and Raji cells, binds to the p40^phoxpromoter, and is required for reporter activity in Raji, but not HeLa cells.

(A) EMSA of nuclear extracts with ³²P-labeledp40^phox PU.1a DNA probe. HeLa (lanes 1-3), HL-60 (lanes 4-6) or Raji (lanes 7-12) nuclear extracts (5 μg) were incubated with the labeled probes alone (lanes 2, 5, and 8) or together with either antibody to PU.1 (lane 10) or a 200-fold molar excess of unlabeled PU.1a (lanes 3, 6, and 9), PU.1b (lane 11), or PU.1c (lane 12) oligonucleotide (see Figure 2B). The specific PU.1-DNA complex (PU.1) and the supershifted complex (SS) are indicated by arrows. Mutational analysis of the −1599 to +104 region of thep40^phox promoter in Raji (B) or HeLa cells (C). The PU.1 sites (open boxes) present in the pGL3-p40-1599 construct were mutated (hatched boxes), and the resultant constructs were assayed for reporter gene activity as in Figure 1. Arrow indicates the reported transcription start site. Data shown are means (± SE) of 3 or more independent experiments.