EMSAs showing the DNA-binding activity of recombinant GST-Z, GST-ZBP-89, and endogenous ZBP-89.

(A) Lane 1, radiolabeled GC oligonucleotide alone; lanes 2 and 3, labeled GC in the presence of GST or GST-Z, respectively. In panels A-C, the arrow indicates the position of the complex. This complex was competed with the unlabeled GC oligonucleotide (lane 4) or oligonucleotide htβ (lane 5). Lane 6 shows the lack of competition by a consensus Sp1 oligonucleotide. (B) Lane 1, radiolabeled GC alone; lanes 2 and 3, labeled GC in the presence of GST or GST-ZBP-89 fusion protein, respectively. The resulting complex was competed with unlabeled GC (lane 4) or with htβ (lane 5); lanes 6 and 7 show the effect of anti–ZBP-89 antibody (lane 6) and preimmune serum (lane 7), respectively, on the ZBP-89 complex. (C) Lane 1, radiolabeled GC alone. In lanes 2 and 3, binding reactions containing uninduced U937 nuclear extract. Lanes 4 to 11 show binding reactions containing PMA-induced U937 nuclear extract. ZBP-89 binding to DNA (lane 4) was competed with a 250-fold molar excess of unlabeled GC (lanes 3 and 5) or htβ (lanes 4 and 6). A consensus Oct-1 oligonucleotide (lane 7) and a consensus Sp1 oligonucleotide were ineffective (lane 10) in competition. Lanes 8, 9, and 11 represent binding reactions preincubated with anti–ZBP-89 antibody (lane 8), preimmune sera (lane 9), and anti-Sp1 antibody (lane 11), respectively.