Fig. 10

Effect of sulfonylureas on the distribution of pp59^Lyn and cAMP-binding ectoprotein, Gce1, between detergent-insoluble glycolipid-enriched rafts (DIGs) and non-DIG structures of the plasma membrane. (140) Isolated rat adipocytes were stimulated with increasing concentrations of different sulfonylureas for 30 min at 30°C. Plasma membranes were prepared by differential centrifugation and then treated with 1% TX-100 for 1 hr on ice. After centrifugation (100,000X g, 15 min), the detergent-insoluble pellet fraction was subjected to sucrose density gradient centrifugation for purification of DIGs. They were recovered from the 8/20%-interface of the gradient, collected by centrifugation and then solubilized in sample buffer. The detergent-soluble supernatant fraction (containing the non-DIG structures) was precipitated and then solubilized in sample buffer. Both DIGs and non-DIGs were analyzed by SDS-PAGE and immunoblotting (IB; chemiluminescence detection) for the presence of pp59^Lyn, caveolin and glucose transporter isoforms (GLUT4) or photoaffinity labeling with N₃-[³²P]cAMP for the presence of Gce1. Phosphorimages and chemiluminescence images of a typical experiment (repeated twice with similar results) are shown. IRS, insulin receptor substrate.