FIG. 6.

(A) Nuclear extracts from LMH/2A cells, with E2 added to the medium 30 min before harvesting, were incubated with [γ-³²P]ATP-labeled DS-ERE probe (Table 1) from the CAV promoter. The ERα-specific antibody ER-Ab10 was added to the nuclear extracts in lane 2, and unlabeled DS-ERE oligonucleotides were added at 50-, 100-, and 200-fold excesses in lanes 3 to 5. Unlabeled competitor oligonucleotides EREcon, nonspecific (Nonsp.), Sp1, and half-site ( site) (Table 1) were added at 50-, 100-, and 200-fold excesses in lanes 6 to 8, 9 to 11, 12 to 14, and 15 to 17, respectively. (B) Nuclear extracts from LMH/2A cells, with E2 added to the medium 30 min before harvesting, were incubated with the [γ-³²P]ATP-labeled EREcon probe in lanes 1 to 4 or with Sp1-DR-NFY-labeled probe from the CAV promoter (Table 1) in lanes 5 to 18. Unlabeled Sp1-DR-NFY oligonucleotides were added at 50-, 100-, and 200-fold excesses as competition with the EREcon probe in lanes 2 to 5. The Sp1-DR-NFY probe was competed with unlabeled Sp1-DR-NFY and EREcon oligonucleotides at 50-, 100-, and 200-fold excesses in lanes 7 to 9 and 10 to 12, respectively. Unlabeled oligonucleotides Sp1, half-site ( site), and nonspecific (Nonsp.) were added at 100- and 200-fold excesses in lanes 13 to 14, 15 to 16, and 17 to 18, respectively. Protein-DNA complexes were resolved on 5% polyacrylamide gels and visualized by autoradiography and are indicated by the numbers 1 to 4 or the letters A to D to the left and right of the gels. FP, unbound free probe.