Binding of Zf9-GST to GC boxes in the uPA promoter.
Binding of Zf9-GST fusion protein to the 32P-labeled uPA GC box oligonucleotide was determined by gel shift assay. Four different mutant oligonucleotides were tested, which contained mutations in one or all of the GC boxes as shown. Sequences of these oligonucleotides are indicated in panel A. Protein-DNA complexes were separated through a 6% polyacrylamide gel and visualized on an image analyzer (panel B). Lane 1, wild uPA GC box alone; lane 2, wild uPA GC box + Zf9-GST; lane 3, wild uPA GC box + Zf9-GST in the presence of a 20-fold excess of unlabeled (cold) oligonucleotide; lane 4, wild uPA GC box + GST; lanes 5-8, mutant uPA GC boxes + Zf9-GST. Lane 5, oligonucleotide with mutations in all 3 GC boxes (abbreviated as “All”); lane 6; mutant oligonucleotide in which 5′ site GC box was mutated (abbreviated as “5′”); lane 7, mutant oligonucleotide in which the middle GC box was mutated (abbreviated as “Mid”); lane 8; mutant oligonucleotide in which 3′ site GC box was mutated (abbreviated as “3′”). The experiment was repeated 3 times with similar results in all experiments; representative results are shown.
