The t-PA –7351C>T enhancer polymorphism affects protein binding affinity. (A) EMSAs were performed following incubation of 32P-labeled wild-type C or mutant T GC-box oligonucleotides with 5 μg nuclear proteins from 4 different human cell lines that are known to express t-PA. (Lanes 1-2) HUVECs; (lanes 3-4) C11STH; (lanes 5-6) HeLa cells; (lanes 7-8) NT2 nuclear extracts; odd lanes (1,3,5,7), wild-type GC-box probe; even lanes (2,4,6,8), mutant GC-box probe. (B-C) Competition and cross-competition EMSA experiments were performed using unlabeled competitor oligomers corresponding to either the C or T allele variant to assess difference in binding affinity of proteins associating with the 2 alleles at the –7351 site. Both wild-type and mutant 32P-labeled GC-box probes were used (B and C, respectively). Arrows to the left indicate the 2 specific complexes. HUVEC nuclear extracts (5 μg) were included in all reactions, except for that in well 1. (B-C, lane 2) Migration patterns produced in the absence of competitor; (lanes 3-6) increasing concentrations of unlabeled oligomers of identical sequence to labeled counterpart (self-competition), 0.1, 1, 10, or 100 ng (approximately 0.5-, 5-, 50-, 500-fold molar excesses); (lanes 7-10) increasing concentrations of the unlabeled variant oligomer (cross-competition of the C and T variant), 0.1, 1, 10, or 100 ng (approximately 0.5-, 5-, 50-, 500-fold molar excesses); (lane 11) 100 ng (500-fold molar excess) of an unlabeled nonspecific competitor. The results indicate a 10-fold greater protein binding affinity to the t-PA –7351C allele compared with the T allele variant.
