Fig. 7. Sphingolipid depletion does not affect the localization of calflagin to the flagellum or DRMs in procyclic T. brucei. (A) Procyclic T. brucei were incubated with 1.5 µM myriocin for 24 hours, and TbSPT2 RNAi cell lines were incubated with or without Tet for 3 days. Bloodstream T. brucei were left untreated or treated with 0.5 µM myriocin for 24 hours. The cells were analyzed by DIC for cell morphology, stained with DAPI for DNA content and analyzed by immunofluorescence using an anti-calflagin antibody to visualize calflagin localization. Calflagin localized to the flagellum under all treatment conditions. (B,C) Procyclic 29-13 and TbSPT2 RNAi lines were treated as described above. Bloodstream T. brucei were left untreated or treated with 0.15 µM myriocin for 24 hours. 2x10⁸ cells were harvested and extracted with 1% ice-cold Triton X-100, loaded on the bottom of an OptiPrep gradient, and ultracentrifuged as described in Materials and Methods. OptiPrep fractions were analyzed by western blotting using calflagin-specific serum. Fractions 2 and 3 represent the buoyant DRM fractions of the cell. Calflagin remains associated with the DRM fractions in both myriocin-treated 29-13 and RNAi +Tet cells as well as the controls, but is lost in bloodforms upon treatment with myriocin.