Figure 10

Total Gαi1/2 associated with PM is increased following overexpression of calnuc-GFP. Light fractions enriched in PM were separated from heavy fractions containing Golgi and ER by sucrose gradient flotation. In parental cells (NT), the Golgi marker (β-COP) and most (84%) of the endogenous calnuc are concentrated in heavy fractions. In cells stably overexpressing calnuc-GFP (CNG), Calnuc-GFP is distributed across both light and heavy fractions, and the % of the total Gαi1/2 in PM-enriched fractions (5–8) is increased from 38% to 63%. Most of the Gαi3 (75%) is found in PM fractions with the remaining (25%) associated with the Golgi in parental cells. Gαi3 has a broader distribution (fractions 1–8) in calnuc-GFP cells (CNG). Postnuclear supernatants in 1.3 M sucrose were overlayed with 1.2 M, 1.14 M, 0.99 M and 0.9 M sucrose, followed by centrifugation at 170,000 × g for 15 h. Eight fractions were collected by centrifugation (100,000 × g for 1 h), and membrane pellets were analyzed by SDS-PAGE followed by immunoblotting. "B" and "C" were plotted as in Figure 6.