PU.I binds to the PU.I site in the EDN intron. (A) PU.I protein was generated in vitro using a rabbit reticulocyte lysate. PU.I (lanes 6 through 10) or unprogrammed control reticulocyte lysate (lanes 1 through 5) was then tested in a bandshift assay for binding to the PU.I site from the EDN intron. Arrows indicate four PU.I/DNA complexes that are competed by excess PU.I oligo (lanes 7 and 8, 10- and 100-fold), but not by excess mutant PU.I oligo (lanes 9 and 10, 10- and 100-fold). Nonspecific complexes formed with control lysate are indicated by open arrows. The free DNA probe is not shown. (B) Nuclear extracts were prepared from HL60 7.7 cells treated for 4 days with BA. These extracts were then tested for PU.I binding activity in a band shift assay. Three protein/DNA complexes are observed (lane 1) that are competed by excess PU.I oligo (lanes 2 and 3, 10- and 100-fold), but not by excess mutant PU.I oligo (lanes 4 and 5, 10- and 100-fold). Preincubation with an anti-PU.I antibody (lane 6), but not with an anti-STAT3 control antibody (lane 7) results in the appearance of two supershifted complexes indicated by arrows. The free DNA probe is not shown.