Figure 4.

Gfi1 and Gfi1b bind to the tandem Gfi1-binding site in vitro. Upper panel: gel-shift analysis was performed using in vitro translated, Myc-tagged proteins and ³²P-labeled double-stranded oligonucleotides as indicated. Where indicated, a 50-fold excess of unlabeled double-stranded oligonucleotides was added as a competitor to show sequence-specific binding. The anti-Myc antibody (9E10) was used to generate a supershift of specific protein–oligonucleotide complexes and to detect the specific Gfi1/Gfi1b-DNA shift complexes labeled 1–4. Only complex 3 is efficiently supershifted by antibody addition and specifically competed by the Gfi1/Gfi1b promoter- and ideal-oligonucleotides, but not by the unrelated CREB-binding site oligonucleotide. Lower panel: sequence similarity analysis comparing the tandem Gfi1-binding site and an ideal Gfi1-binding site used for the gel-shift analysis.