Characterization of the ZBP-89–binding site within theCD11b gene.

(A) Sequence of the double- or single-stranded oligonucleotides used in EMSA are shown. Mutations incorporated into the GC1 oligonucleotide are shown in lowercase letters, whereas wild-type nucleotides are represented by dashed lines. (B) Lane 1, radiolabeled GC1; lanes 2 to 9, binding reaction with GST-Z before (lane 2) or after competing with the following oligonucleotides: unlabeled GC1 (lane 3), M1 (lane 4), M2 (lane 5), M3 (lane 6), M4 (lane 7), M5 (lane 8), M6 (lane 9). (C) Same as in panel B except that GST-ZBP-89 was used instead of GST-Z. (D) Competition analysis of GC1 binding to GST-Z using mutant oligonucleotides. Lane 1, radiolabeled GC1 alone. Lane 2, binding of GC1 to GST-Z. Competition by the M4b-e oligonucleotides (lanes 6, 7, 8, and 9) was approximately equivalent to that of GC1 (lane 3), whereas competition by M4 (lane 4) and M4a (lane 5) was minimal. (E) Binding of the purine-rich single-stranded GC1/for oligonucleotide to MS-2. Lane 1, radiolabeled GC1/for single-stranded oligonucleotide alone; lane 2, the MS-2 complex produced by mixing the labeled GC1/for oligonucleotide with nuclear extract from U937 cells treated with PMA for 24 hours; lanes 3 and 4, competition in the presence of unlabeled GC1/for (lane 3) or M4a/for (lane 4). Lanes 5 to 7, lack of binding of recombinant GST-ZBP-89 to radiolabeled GC1/for in the absence (lane 5) or presence of unlabeled GC1/for (lane 6) or unlabeled M4a/for (lane 7).