GGAA element −65 specifically binds PU.1. (A) An oligonucleotide encompassing the Ets-binding site −65 was used in a band shift assay with nuclear extracts of HL-60 cells. One prominent and several minor complexes are observed (lane 1). All complexes can be competed with a 10- to 100-fold molar excess of cold self oligo (lanes 2 and 3), but not with mutant oligo (lanes 4 and 5), indicating that the complexes bind specifically. Binding is also inhibited by addition of the PU.1 binding element of the G-CSFR and by E4TF1, a sequence that binds GABP, but not by element −45 (lanes 6 through 11). (B) To identify the protein(s) binding to −65, 2 μg of specific antibody against different Ets family members was added before the addition of the probe. Anti-ATF3 was used as a negative control. Complexes are only supershifted when anti-PU.1 is added (lane 5). The arrow indicates a nonspecific complex that appears after addition of anti-GABP (lane 6). However, this complex is also observed when no nuclear extract is added (data not shown). These results show that PU.1 binds to element −65. Similar results were obtained when using U937 cells (data not shown).