Fig. 3. Effect of cholesterol depletion on DRM association of NPP3. MDCK cells stably transfected with NPP3-WT cDNA were labeled with [³H]cholesterol for 20 hours, then treated (MCD) or mock-treated (mock) with 10 mM methyl--cyclodextrin for 30 minutes before detergent extraction. (A,B) Distribution of NPP3 along the sucrose gradient fractions after solubilization with Lubrol WX (A) or Triton X-100 (B). Cell homogenates were detergent-extracted and separated on sucrose gradients and the distribution of NPP3 was analyzed as in Fig. 2. The bar charts show quantification of the DRM-associated (fractions 3-6; DRM) and soluble proteins (fractions 7-12; Sol). (C) Cholesterol content of Lubrol WX and Triton X-100 DRMs. The amount of cholesterol in the gradient fractions was quantified by counting the radioactivity of pooled aliquots of the DRM and soluble fractions by liquid scintillation.