Fig. 5.

Interaction between ERα and EGFR/c-Src in the aromatase activity induction. A, MCF-7 cells were not transfected (-) or transfected with c-Src RNAi and then transfected with His₆-arom vector. Aromatase protein was purified using Ni-NTA agarose beads after which the complexes were resolved in SDS-PAGE. In another set of experiments the same amount of cell lysate was immunoprecipitated (IP) with ERα antibody. Immunoblotting was performed using the anti-ERα, anti-EGFR, anti-c-Src, and antiaromatase antibodies. B, MCF-7 were pretreated with 10 μm AG1478 or 1 μm ICI for 30 min and then exposed to 1 nm E₂ or 100 ng/ml EGF. After 10 min, aromatase activity was performed. The values represent the means ± se from triplicate assays. *, P < 0.01 compared with vehicle; **, P < 0.01 compared with E₂- or EGF-treated samples. C, MCF-7 cells transiently transfected with His₆-arom were pretreated with or without 10 μm AG1478 or 1 μm ICI for 30 min and then exposed to 1 nm E₂ or 100 ng/ml EGF for 10 min. The membrane was probed with antiphosphotyrosine (pTyr) antibody. To verify equal loading, the membrane was probed with antiaromatase antibody. P, Microsomal extracts from placenta. As negative controls we used the supernatant removed after incubation with Ni-NTA agarose beads (S) and vector-transfected MCF-7 cell lysates incubated with Ni-NTA agarose beads (NC). The histograms represent the means ± se of three separate experiments in which band intensities were evaluated in terms of OD arbitrary units and expressed as percentages of the control, which was assumed to be 100%. *, P < 0.01 compared with vehicle; **, P < 0.01 compared with E₂- or EGF-treated samples. kD, Kilodaltons.