GABP from U937 myeloid nuclear extract binds to the mNE promoter. (A) A double-stranded oligonucleotide probe corresponding to -95/-71 of the mNE promoter was radiolabeled with 32P-dCTP. EMSA was performed with 10,000 cpm probe and 10 μg U937 myeloid nuclear extract. A species from U937 cells bound to this probe (lane 1, filled arrow) and was competed for by a 100-fold molar excess of homologous unlabeled probe (lane 2) but not by a 100-fold excess of irrelevant probe (lane 3). Binding of this species was abrogated by antibody to either GABPα or GABPβ (lanes 4 and 5, respectively) but not by preimmune serum (lane 6). Binding by PU.1 (open arrow) and a proteolytic degradation product of PU.1 (asterisk) was also seen. (B) EMSA was performed with the radiolabeled -95/-71 mNE probe and U937 nuclear extract. Antibody to PU.1 abrogated binding by PU.1 (lane 3), and this effect was eliminated by inclusion of PU.1 peptide in the binding reaction (lane 4); preimmune serum had no effect on its binding (lane 5).
