Fig 5. Flotillins recruit ³H-Sph to specific cellular membrane domains.

A. Detergent resistant membrane (DRM) isolation by sucrose gradient centrifugation of ³H-Sph labelled WT and Flot1^-/- primary MEFs. ³H-Sph levels in fractions from the gradient were analysed by liquid scintillation counting. Data are means with SEM, N = 3 separate experiments. Western blots below the graph shows caveolin 1 distribution in the gradient fractions. Full scans of all blots are shown in S3 Data File. B. ³H-cholesterol distribution into DRMs in WT and Flot1^-/- MEFs. Data are means with SEM. Western blots below the graph show flotillin 1 distribution in the gradient fractions. C. Overexpression of flotillins increases Sph accumulation in HeLa cells. HeLa cells were transfected with GFP-tagged proteins and the overexpressing cells were FACS sorted and incubated with ³H-Sph. Radioactivity was measured with liquid scintillation. Data are means with SEM, N = 3 separate experiments. One way ANOVA with Dunnett’s multiple comparisons test, Flot-GFP expressing cells compared to all other groups. D. Ectopic re-localisation of flotillins to mitochondria. Flotillin-1-GFP is co-expressed with either flotillin 2 or flot2-mito, the latter two constructs lack a fluorescent tag. Mitochondria are visualised using mitotracker dye, the lower panels show magnified views of both fluorescence channels within the boxed areas. E. Quantification of ³H-Sph incorporation into mitochondria. Cells were loaded with ³H-Sph for 5min before isolation of mitochondria, and counting of radioactivity in the isolated mitochondria by liquid scintillation. Bars are means and SD, N = 5, Student’s t-test.