Fig. 2. Dictyostelium DdRab11 is associated with contractile vacuole membranes.(A,B) Post-nuclear extracts prepared from homogenized cells were fractionated on Percoll gradients as described in Materials and Methods. After fractionation, samples were subjected to SDS-PAGE followed by western blot analysis using antibodies to DdRab11 and RabD. DdRab11 protein was confined to the top fractions of the gradient that corresponded to non-lysosomal membranes vesicles. Bottom fractions represent lysosomal membrane fraction that contained lysosomal marker enzymes. (C,D) Protein samples (20 µg) from post-nuclear supernatant (PNS), purified contractile vacuole (light membranes) and purified lysosomes were subjected to SDS-PAGE and western blot analysis. DdRab11 and RabD were both enriched in contractile vacuole membranes, but only RabD was enriched in purified lysosomes.