Fig. 1.

Subcellular fractionation by equilibrium density gradient centrifugation does not separate different endomembranes. (A) Gradient distributions of late endosomal proteins syntaxin-8 (1:1000) and Rab9 (1:500), lysosomal prenylcysteine lyase (1:1000), Golgi-localized Vps45 (1:500), and TGN-targeted TGN38 (1:1000) were determined by immunoblotting equal volumes of the gradient fractions. (B) Distribution of cholesterol as measured using the Amplex red cholesterol assay. Antibody dilutions used for immunoblotting are in parentheses. TGN, trans-Golgi network.