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. 2009 Sep 1;284(44):30187–30199. doi: 10.1074/jbc.M109.026948

FIGURE 3.

FIGURE 3.

The G5 string in G1HE-(201–235) is required for ZBP-89 binding. A, binding of the recombinant ZBP-89 DNA binding domain (amino acids 169–281) GST fusion protein (GST-ZBP-89 DBD) to the G1HE-(201–235) region. One μg of recombinant protein was incubated with probe. Note that binding of GST-ZBP-89 DBD to the probe was markedly reduced when one deoxyguanosine residue was deleted in G1HE-(215–219) (G1HE 201–235 G4). A known ZBP-89 binding sequence from the mouse p21waf1 promoter and its mutant (p21 and p21 mut, respectively) (35) were used as controls. The sequences of the p21waf1 and p21waf1 mut oligonucleotides are given under “Experimental Procedures.” B, binding of GST-ZBP-89 DBD to the G1HE-(201–235) probe either in the absence or presence of unlabeled competitors. G1HE-(201–235), G1HE-(201–235) G4, p21waf1, and p21waf1 mut competitors were added to the binding reactions in the amounts indicated. C, endogenous ZBP-89 binding to G1HE-(201–235) was detected by EMSA supershift assays in K562 nuclear extracts. K562 nuclear extracts (3 or 6 μg) were incubated with the G1HE-(201–235) probe either in the absence or presence of competitors or antibodies. A 100-fold molar excess of unlabeled G1HE-(201–235) or G1HE-(201–235) G4 was added in lanes 1 and 2, respectively. Antibodies for ZBP-89 (lanes 4, 5, and 9), Sp1 (lanes 6 and 10), and Sp3 (lanes 7 and 11) were added to determine supershifted complexes (S). DNA-protein complexes are indicated by 1, 2, and 3.