Fig. 3. Inhibition of cell attachment by overexpressed PH-GFP chimeras in COS-7 cells. COS-7 cells were transfected with the indicated PH-GFP constructs for 1 day. Cells were removed by mild trypsinization and divided into three aliquots. One aliquot was centrifuged and the cells were immediately lysed in Laemmli buffer (labeled c). The other two aliquots were plated into 35mm culture dishes and the cells were allowed to attach for 30 minutes (labeled a). After this, the medium was removed and the cells were washed twice with 2 ml of ice-cold Dulbecco's PBS before lysis in the same volume of Laemmli buffer that was added to the first aliquot of cells. Samples were then sonicated (but not boiled) before separation by SDS-PAGE. Electrophoresis gels were analyzed in a Storm 860 Phosphorimager (Molecular Dynamics) using the blue fluorescent laser for quantitation of the GFP fusion protein band in the gel. The fraction of GFP signal found in the attached cells relative to the total amount of cells seeded was calculated for each construct including GFP alone, which served as a control. Representative gel samples are shown in (A), and the mean±s.e.m. from five experiments performed in duplicates are shown in (B).