Fig. 4

Influence of anti-ER antibodies on the DNA-protein complex formation in MCF-7 and 21PT cells. MCF-7 and 21PT cells were grown in complete medium as described in the legend to Figure 3. Nuclear extracts were prepared, and protein content was measured and subjected to EMSA as described in Figure 3. (A) The structural and functional domains of ER. The epitope maps of the three ER-antibodies used in this experiment are shown below the sketch, details of the ER-antibodies are described in the Materials and Methods. (B) The influence of ER-Ab-1, mouse IgG, and anti-NF-κ-p50 antibody (rabbit polyclonal IgG raised against 15 amino acid residues mapping at the NLS region of NF-κB-p50, mouse, rat, and human reactive, Santa Cruz Biotechnology, Santa Cruz, CA). Binding reaction conditions were the same as described above. Nuclear extracts of 2.5 µg or 0.5 µg of either of these antibodies or mouse IgG, and ³²P-ERE-Oligo (30,000 cpm, 1 ng) were used in 10 µl of duplicate binding reactions and incubated for 30 min at room temperature followed by EMSA analysis. The retarded DNA-protein bands are indicated by the arrows. (C) The similar analysis of duplicate samples of binding reactions with nuclear extracts (2.5 µg) of MCF-7 and 21PT cells in the presence of 0.5 µg of anti ER-antibodies TE1-11 or SRA 1010 under the conditions described above. These experiments were repeated three times with a different order of additions and preincubations of nuclear extracts, either with antibodies followed by addition of ³²P-ERE-oligo or with ³²P-ERE oligo followed by addition of ER-antibody. All of these experiments showed results similar to those presented here.