GGAA-element −45 binds an unidentified protein. (A) An oligonucleotide encompassing the Ets-binding site −45 was used in a band shift assay with nuclear extracts of HL-60 cells. Five complexes (C1 through C5) bound to −45 (lane 1), of which C2, C3, and C4 could be competed with excess of self oligo (lanes 2 and 3). Complexes C1 and C5 were nonspecific, because these complexes could not be competed. Moreover, C2 and C3 were also nonspecific, because they could be competed with mutant oligo (lanes 4 and 5). With supershift analysis it was shown that C4 contains PU.1 (not shown), explaining why binding of C4 was blocked by the addition of −45, E4TF1, −65, and PU.1 oligonucleotides. Similar results were obtained when using U937 cells (data not shown). (B) Longer exposure of the gel shown in (A). Binding of two complexes with low mobility (C6 and C7) was detected. Binding of these complexes could be inhibited with excess of self oligo only, showing the binding specificity of these complexes. These complexes could not be supershifted with antibodies against Ets-1, Ets-2, Fli-1, PU.1, and GABP. Similar results were obtained when using U937 cells (data not shown).