Fig. 5. Inhibition of EGF-stimulated InsP formation by overexpressed PH domain chimeras in COS-7 cells. COS-7 cells were transfected with cDNA encoding the human EGF receptor, together with selected GFP-PH domain fusion constructs as described under Materials and Methods. One day after transfection, cells were labeled with myo-[³H]inositol for 24 hours. Cells were stimulated by EGF for 30 minutes in the presence of 10 mM LiCl, and [³H]-labeled inositol phosphates were separated by Dowex minicolumns and measured by liquid scintillation counting. The relatively small activation of PLC by EGF in these cells did not permit direct analysis of the 1,4,5-isomer of InsP₃ and the pooled InsP₃ and InsP₂ samples were used as an indicator of overall PLC activity. The InsP response of the cells was normalized to the value observed after EGF stimulation of cells expressing only GFP, which was about a twofold increase in average. The PI 3-kinase inhibitor wortmannin (wm) was added 10 minutes before EGF treatment. Mean±s.e.m. of 3-5 experiments are shown performed in duplicate (except for Akt-PH, where n=2).