Mapping of transcription factor binding sites in thehEPCRgene promoter by EMSA. Double-stranded oligonucleotides, corresponding to active regions identified in 5′ deletion analysis, were labeled with α-³²P, incubated with EA.hy926 cell nuclear extracts, and separated by EMSA using native-polyacrylamide gel electrophoresis (PAGE; see “Materials and methods”). The probes used were as follows: (panel A) RI; (panel B) RII; (panel C) RIII; (panel D) RIV; and (panel E) RV (Table 2). Where indicated, an excess of unlabeled oligonucleotides, containing mutated bases within putative transcription factor binding sites, was included during incubation (Table 2). For supershift analysis, 100 μg/mL polyclonal antibodies to Sp1 or Sp3 was included as indicated. The arrow in panel D indicates the presence of a supershifted band (con = consensus, mt = mutant).