Figure 5.

OptiPrep gradient fractionation of detergent-free plasma membranes from MDCK epithelial or S49 lymphoma cells. Sonicated plasma membranes were brought to 23% OptiPrep in 4 ml and were placed at the bottom of the tube. A linear gradient of 20–10% OptiPrep was poured on top of the plasma membranes. After centrifugation, either 1 ml (panels A, B, C, and F) or 0.7-ml fractions (panels D and E) were taken from the top of the tube. These fractions were analyzed for total protein content by Bradford assay (panels A and D), G protein or caveolin content by Western immunoblotting (panels B, C, and E), or adenylyl cyclase activity (panel F). The specific activity of isoproterenol- (Iso-) and forskolin- (Fsk-) stimulated adenylyl cyclase in the individual fractions 1–6 and combined fractions 7–11 are shown in panel F. Antibody preparations utilized for immunoblotting were the same concentration as Figure 4 but, in addition, 584 antiserum was employed for detection of long and short isoforms of α_s at a dilution of 1:5,000.