Figure 1

Localization of endogenous Gβ5/RGS7 complexes to lipid rafts in PC12 cells. A. Gβ5/RGS7/R7BP proteins are expressed endogenously in PC12 cells. Hela and PC12 cells were lysed and samples were loaded in SDS-PAGE gels and subjected to immunoblotting using anti-R7BP (N-terminal), anti-RGS7, anti-Gβ5, and actin antibodies as described in Methods. The relative mobility of the major immunoreactive bands (in kDa) is indicated on the right. B. Determination of DRM (lipid raft) and non-DRM markers along a sucrose density gradient in 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, LAT and PSD-95 as markers 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. C. Localization of endogenous Gβ5 and RGS7 proteins in PC12 cells. PC12 cells were lysed, and subjected to a sucrose density gradient. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the endogenous Gβ5 and RGS7 proteins as indicated. D. Densitometric quantification of the distribution of endogenous Gβ5 and RGS7 proteins among DRM and non-DRM fractions in sucrose density gradients. Immunoblots of sucrose density gradients such as that shown in C were analyzed by densitometry and the distribution of the indicated immunoreactivity between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblots, with error bars representing the S.E.M. The results shown are representative of three experiments completed with similar results.