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Fig. 4. Inhibition of cell spreading by overexpressed PH domain chimeras in COS-7 cells. COS-7 cells transfected with the indicated PH-GFP constructs for 24 hours were removed from the culture dishes with gentle trypsinization and re-plated onto fibronectin-coated glass cover slips as detailed under Materials and Methods. After 10 minutes, cells were fixed and stained with TRITC-phalloidin. Cells overexpressing the GFP fusion proteins were identified by fluorescent microscopy and were classified into three groups: unspread (adherent with no projections), partially spread (adherent with limited lamellipodia) and fully spread. The percentage of fully spread cells was then calculated for each group of cells expressing the GFP fusion proteins and related to the untransfected controls. When added, the PI 3-kinase inhibitor, LY 424002 (Ly) was added 10 minutes before plating on fibronectin. Mean±s.e.m. of 3-6 experiments are shown.





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