FIGURE 6.

Ets2 and c-Myc interact in vitro and in cultured human breast cancer cells. A, co-immunoprecipitation of Ets2 with c-Myc. MCF-7 cell lysates were incubated with normal IgG or anti-c-Myc antibody for 60 min at room temperature before capturing the c-Myc immune complex using protein A-coated Sepharose beads. Precipitated proteins were determined by immunoblotting (IB) using the antibodies as indicated. B, co-immunoprecipitation (IP) of c-Myc with Ets2. Immunoprecipitation was conducted using anti-Ets2 and other Ets antibodies as indicated in reverse order to that shown in A. Co-immunoprecipitation of c-Myc with Ets2 was determined by immunoblotting. Data are representatives of one of three similar experiments. C, in vitro binding between Ets2 and c-Myc by GST pulldown assay. GST, GST-Ets1, and GST-Ets2 purified on glutathione beads were incubated with the nuclear fraction or total cell lysates of MCF-7 cells, which was followed by centrifugation, stringent wash, and subsequent protein analysis in the precipitates by Western blotting. Immunoreactivity of c-Myc and GST proteins was revealed using specific anti-myc and anti-GST antibodies. Results were from one of three similar experimental results. D, quantification of c-Myc binding to GST fusion proteins by densitometry. Results are mean ± S.D. of detected c-Myc normalized by GST proteins as ratios from three similar experiments. Asterisks indicate significant differences in comparison with GST-Ets1 or GST only with p < 0.001.