Figure 4

Binding of ER to the identified ERE-1 in the GFBP-6 gene (EMSA). Nuclear extracts from SaOS-2 cells were incubated with a ³²P-labeled probe harboring the ERE-1 (Lane 1-5) of the IGFBP-6 gene. The resulting complexes were resolved using non-denaturing PAGE. A labeled mutant-type ERE-1 probe was used as a negative control (Lane 6). For competition EMSA, 50-fold and 100-fold excesses of the unlabeled probe harboring the wild-type ERE-1 (Lane 4, 5) of the IGFBP-6 gene promoter were added during the pre-incubation period. Responsive banding (see arrow in Figure 4) of the wild-type ERE-1 binding complex was observed. Addition of a cold consensus ERE-1 (Lane 4, 5) decreased or eliminated the formation of specific complexes. The retarded band of the mutant-type ERE-1 binding complex was not observed when labeled mutant-type ERE-1 was incubated with nuclear extracts (Lanes 6). The ERα antibody did bind to the putative ERE-1-ER complexes. These specific complexes (Lane 3) were delayed and formed a supershifted band, compared with the ERE-1-ER complexes (Lane 2). The arrow indicates ER binding.