Fig. 5. Oestradiol-induced assembly of Src–PI3-kinase–ERα complex. Quiescent MCF-7 cells were left unstimulated or stimulated for 5 min with 10 nM oestradiol. Lysates were immunoprecipitated with either anti-p85 (A), or anti-Src (B) or anti-ER (C) antibodies. Immunocomplexes were analysed by immunoblot with antibodies against the indicated proteins. (D) Either GST–Src (left section) or GST–HEG14 (right section) was incubated in the absence or presence of 10 nM oestradiol with the indicated ³⁵S-labelled proteins. In vitro interaction with GST fusion proteins was performed as described in Materials and methods. Proteins were eluted by Laemmli sample buffer, resolved on SDS–PAGE, then finally revealed by autoradiography.