Figure 4

ERα binding to EREs detected in the MUC1 promoter. The EMSA of the complexes (C1, C2 and C3) developed by ERα from T47D nuclear lysate and ³²P-labeled oligonucleotides containing the MUC1 promoter EREs (for details see "Materials and Methods"). A: Binding of ERα to ³²P-labeled oligonucleotides ERE1, ERE2, ERE3, ERE4 and ERE5, containing 1/2 consensus core sequence of the classical ERE, and to palindrome like ERE (putative ERE-6 and Vit ERE). Lanes 1, 3, 5, 7, 10, 12, 19 and 26 – complexes developed in absence of anti-ERα Ab; lanes 2, 4, 6, 8, 11, 13, 20 and 27 – partial or complete inhibition of complex formation by anti-ERα Ab; lanes 14, 21 and 28 – binding of ERα to ³²P-ERE2, Vit-ERE and ERE6, respectively, in the presence of "cold" Oct1 oligonucleotide; lanes 15–18 – binding of ERα to ³²P-ERE2 in presence of decreasing amount of the "cold" ERE2; lanes 22–25 – binding of ERα to ³²P-Vit ERE in presence of decreasing amount of the "cold" Vit ERE (* indicates an intermediate complex developed when binding reaction was kept on ice); lanes 29–32 – binding of ERα to ³²P-ERE6 in presence of decreasing amount of the "cold" ERE6. B: Effects of anti-ERα Ab and normal rabbit serum (NRS) on ERα binding to MUC1 specific and mutated EREs. Lanes 1, 6 and 11 – binding of ERα in absence of anti-ERα Ab; lanes 2, 7 and 12 – partial or complete inhibition of binding complexes by anti-ERα Ab; lanes 3, 8 and 13 – binding of ERα in presence of NRS; lanes 4, 9 and 14 – binding of ERα to mutated ERE in absence of anti-ERα Ab; lanes 5, 10 and 15 – binding of ERα to mutated ERE in presence of anti-ERα Ab.