Fig. 4. Reciprocal regulation of PI3-kinase and Src activities stimulated by oestradiol. (A and B) Quiescent MCF-7 were untreated or treated with 10 nM oestradiol for 5 min in the absence or presence of the indicated inhibitors. LY294002 (20 µM) and PP1 (10 µM) were added to the cell medium 10 min before hormone stimulation. In panel A lysates were analysed for P-Akt (left section). The same filter was re-probed with anti-Akt antibody (right section). In panel B PI3-kinase activity of p85 immunocomplexes was assayed and radiolabelled phospholipids revealed by autoradiography. (C) Quiescent MCF-7 were untreated or treated with 10 nM oestradiol for 3 min in the absence or presence of 20 µM LY294002. Two different times of pre-incubation with the inhibitor (10 and 30 min) were used. Src activity of Src immunocomplexes (right half four lanes) was assayed using acidified enolase (en) as a substrate. Left half four lanes show Src activity of proteins immunoprecipitated by control antibodies. (D) NIH 3T3 fibroblasts were transfected with ERα expressing plasmid alone or together with the Δp85α construct. Cells were made quiescent, then left untreated or treated for 3 min with 10 nM oestradiol. In the upper section: lysates were immunoprecipitated with anti-Src antibody and Src activity was assayed. In the lower section: lysates were analysed for either p85 or ERα expression by immunoblot.