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. 2009 Jan 21;296(3):C607–C619. doi: 10.1152/ajpcell.00488.2008

Fig. 5.

Fig. 5.

Disruption of lipid rafts ablates the regulation by CCE of the N-glycosylation-defective mutant of AC8. A: in vivo cAMP accumulation on disruption of lipid rafts by cholesterol extraction (methyl-β-cyclodextrin; MβCD) followed by cholesterol replenishment (cholesterol-MβCD complexes). HEK-293 cells transiently transfected with either WT or triple N-glycosylation-defective mutant of AC8 (AC8N814Q/N818Q/N855E) were incubated with 2-[3H]adenine for 45 min, followed by incubation for 45 min with both 2-[3H]adenine and 10 mM MβCD in medium without serum, which was followed, or not, by incubation with cholesterol-MβCD complexes for 45 min (all assays were ±4 mM extracellular Ca2+; see Fig. 2). Data represent means ± SE of at least 3 separate experiments performed in triplicate. Conditions were compared using two-tailed Student's t-test. P < 0.05 was considered significant (***P < 0.0005, **P < 0.005, *P < 0.05; ns, not significant). Statistical significances drawn within bars are relative to their own control (i.e., without MβCD). Inset: in vivo cAMP accumulation on disruption of lipid rafts by sphingomyelinase treatment. Shown are means ± SE of one representative experiment. B: fractionation of raft and non-raft membranes on disruption of lipid rafts by cholesterol extraction. HEK-293 cells stably expressing WT AC8 were incubated for 1 h in medium without serum ±10 mM MβCD. Raft and non-raft membranes were then prepared (see experimental procedures). Aliquots from each fraction were probed for AC8 and caveolin by SDS-PAGE followed by immunoblotting. Signals from fractions 2 and 7 are shown, representative of raft and non-raft membranes, respectively. Fraction 2: 7.5–23% sucrose (top to bottom of the fraction); fraction 7: 33–39.5% sucrose (top to bottom of the fraction). CTR, control. C: measurement of intracellular Ca2+ concentration was performed as indicated in experimental procedures in HEK-293 cells treated with and without 10 mM MβCD for 1 h in medium without serum. Application of 100 nM TG and 4 mM extracellular Ca2+ is indicated by arrows.