Figure 5

Effect of R7BP on the distribution of the endogenous Gβ5/RGS7 complex among detergent-resistant membrane (DRM) and non-DRM fractions in PC12 cells. A. Determination of DRM (lipid raft) and non-DRM markers along a sucrose density gradient in vector-transfected PC12 cells. The DRM (lipid rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation as detailed in Methods. Fractions were analyzed by immunoblotting for flotillin-1 as a marker for DRM and the β2 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 endogenous Gβ5 and RGS7 proteins in control PC12 cells transfected with empty pcDNA3-HA vector (vector only). PC12 cells were transfected with empty vector, 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 endogenous 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 endogenous Gβ5 and RGS7 proteins in PC12 cells expressing wild type (wt) HA-R7BP. PC12 cells were transfected with wt HA-R7BP and analyzed as indicated in the legend to B. D. Localization of endogenous Gβ5 and RGS7 proteins in PC12 cells expressing the non-palmitoylated HA-R7BP-SS mutant. PC12 were transfected with HA-R7BP-SS and analyzed as indicated in the legend to B. These results are representative of three total such experiments performed with similar findings.