Figure 6

Effect of R7BP on the distribution of the Gβ5/RGS7 complex among detergent-resistant membrane (DRM) and non-DRM fractions in HEK-293 cells. A. Determination of endogenous DRM (lipid raft) and non-DRM markers along a sucrose density gradient in HEK-293 cells. The DRM (lipid rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation. Fractions were analyzed by immunoblotting for Fyn as a marker for DRM and the α1 subunit of Na-K ATPase as a non-DRM marker. Fractions 1–5 were enriched in the DRM marker, while fractions 6–10 carried the non-DRM marker. B. Localization of transfected AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were transfected with the indicated constructs, lysed and subjected to a sucrose density gradient centrifugation. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the transfected Gβ5 and RGS7 proteins as indicated. Immunoblots of the sucrose density gradients shown were analyzed by densitometry and the distribution of the indicated immunoreactive bands between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblot, with error bars representing the S.E.M. C. Localization of co-transfected wild type HA-R7BP, AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were co-transfected as indicted and analyzed as indicated in the legend to panel B. D. Localization of co-transfected HA-R7BP-SS, AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were co-transfected as indicated and analyzed as indicated in the legend to panel B. These results are representative of four total such experiments performed with similar findings.