FIG. 5.

(A) EMSA reaction with the [γ-³²P]ATP-labeled ERE consensus oligonucleotides (Table 1) (EREcon) as the probe and nuclear extracts from LMH cells (lane 1) or LMH/2A cells (lanes 2 to 6). Nuclear extracts used in lanes 2 were harvested from LMH/2A cells grown in complete medium without E2 supplementation, and E2 was added to the EMSA binding reaction for lane 3. Nuclear extracts used in lanes 4 to 6 were from LMH/2A cells grown in complete medium supplemented with 10⁻⁷ M E2 for 30 min before harvest. The ERα-specific antibody ER-Ab10 was added to the nuclear extracts in lane 6 and 7. a, E2 added to the EMSA binding reaction; b, nuclear extracts from cells grown in medium supplemented with 10⁻⁷ M E2 30 min prior to harvesting for nuclear extracts; +, present; −, absent. (B) Nuclear extracts from LMH/2A cells with E2 added to the medium 30 min before harvesting were incubated with the EREcon probe. Lane 1 has no nuclear extracts added as a negative control, ER-AB10 was added to lane 3, and unlabeled EREcon oligonucleotides were added at 50-, 100-, and 200-fold excesses in lanes 4 to 6. Unlabeled oligonucleotides from the CAV promoter, DS-ERE, and DR-15 (Table 1) were added at 50-, 100-, and 200-fold excesses in lanes 7 to 9 and 13 to 15, respectively. Unlabeled nonspecific and Sp1 consensus oligonucleotides (Table 1) (Nonsp and Sp1) were added at 50-, 100-, and 200-fold excesses in lanes 10 to 12 and 16 to 18, respectively, and an unlabeled single half-site response element (Table 1) ([1/2] site) was added at 100- and 200-fold excesses in lanes 19 to 20. Protein-DNA complexes were resolved on 5% polyacrylamide gels and visualized by autoradiography. Protein-DNA complexes 1 to 4 are indicated to the left of the gels. FP, unbound free probe; SS, supershifted band.