Figure 8.

EGF and HRGβ stimulation of MDA-MB-435 cells induces recruitment of the p85 subunit of PI 3-K to the phosphotyrosine cellular fraction. Cells were serum starved for 24 h and harvested as previously described. Cells (7.5 × 10⁶ per sample) were incubated in the absence or presence of 100 ng/ml EGF (A) or 100 ng/ml HRGβ (B) for the indicated periods at 37°C. Cells were then lysed, and phosphotyrosine-containing proteins were immunoprecipitated with the anti-phosphotyrosine mAb PY20 coupled to protein A-Sepharose beads. Washed immunocomplexes were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blotting was performed on membranes with the anti-phosphotyrosine mAb 4G10 to detect phosphotyrosine-containing proteins (upper panels). Blots were stripped and reprobed for EGFR or erbB3 (A and B, respectively; our unpublished data) and for p85 (lower panels). Similar data were obtained in at least two additional assays.