Functional significance of mapped transcription factor binding sites. The function of each mapped transcription factor binding site was investigated by introducing an appropriate mutation into the construct SEAP.–518 or SEAP.–227. These mutations are represented schematically on the left side of the figure. All of the identified binding sites within the wild-type constructs SEAP.–518, SEAP.–265, SEAP.–227, and SEAP.–137 are shown. The Sp1 family protein binding sites are represented by gray circles, and the NF-1 binding site is represented by a pale gray rectangle. The overlapping circles represent the cluster of 3 overlapping Sp1 sites, all of which were mutated for this investigation. Symbol(s) that are crossed represent those site(s) mutated in each construct. The precise mutation(s) introduced were those indicated in Table 2. Mutant constructs (named according to the corresponding EMSA in Figure 6) were transiently transfected into EA.hy926 cells (▪) or HUVECs (▦) as described in “Materials and methods.” Normalized reporter activity is shown relative to the wild-type SEAP.–518 construct ± standard error. The log activity of each mutant was compared with this using a paired t test; those with significantly different activities (P < .05) are indicated with an asterisk.