{"SUPP CAPTION FIGS1.png": "'\\n\\n**Figure S1 \\\\(|\\\\) Localisation of GRAF1 to tubular membrane structures is temperature sensitive and dependent on the BAR and PH domains.****A,** Western blots showing the different forms of GRAF1 detected in adult rat brain and their differential presence/absence in cultured SH-SY5Y (human neuroblastoma), HeLa (human fibroblast), K562 (human Chronic Myeloid Leukaemia), and MEF (mouse embryonic fibroblast), cells. Western blots of purified GRAF1 and myc-tagged GRAF1 (from lysates of HeLa cells overexpressing this protein) are shown for comparison. **B,** Confocal micrograph of an NIH 3T3 cell stained for endogenous GRAF1 distribution. **C,** Confocal micrographs of HeLa cells fixed either at 4degC or 37degC for 10 minutes in 4% paraformaldehyde and then stained for endogenous GRAF1. Note the absence of GRAF1-positive tubules in the 4degC fixation image. **D**, Confocal micrographs showing the tubular localization of overexpressed myc-tagged GRAF1 BAR+PH protein in HeLa cells and the cytoplasmic localization of a similarly overexpressed protein with a BAR domain mutation (KK131/132EE). **E,** Confocal micrograph showing the cytoplasmic and punctate localization of overexpressed myc-tagged GRAF1 BAR protein in HeLa cells. **Scale bars = 10\\\\(\\\\mu\\\\)m.**'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Figure S5 \\\\(|\\\\) Nature of GRAF1-positive endocytic structures.****A and B,** Confocal micrographs of HeLa cells overexpressing GFP-tagged flotillin1 (A) or GFP-tagged caveolin1 (B) and co-stained for endogenous GRAF1. Note the lack of colocalization. **C and D,** Confocal micrographs of HeLa cells overexpressing myc-tagged GRAF1 BAR+PH and flotillin1 (E) or caveolin1 (F) incubated with CTxB for 5 minutes. Note the colocalization of GRAF1 BAR+PH and flotillin1 in CTxB-positive tubular structures. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "SUPP CAPTION FIGS4.png": "'\\n\\n**Figure S4 \\\\(|\\\\)****GRAF1 BAR+PH overexpression affects CTxB uptake but not transferrin uptake.****A and B,** Confocal micrographs of HeLa cells transfected with myc-tagged GRAF1 BAR+PH and incubated with CTxB or transferrin for 15 minutes before fixation and staining. **C,** The graph shows the quantification of images such as depicted in (A). Cells were scored for expression of GRAF1 BAR+PH (over a threshold corresponding to maximum autofluorescence) and CTXB internalization (over an arbitrarily-set threshold above background). Note the reduction of the number of transfected cells internalising CTxB compared with controls. **D,** Live cell microscopy of NIH 3T3 cells expressing GFP-tagged GRAF1 and incubated with CTXB and transferrin (Tfn) at 4degC before chasing their internalization from the time of warming to 37degC (time=00:00). Note the internalising GRAF1-positive tubule containing CTxB. Note also the lack of colocalization of GRAF1-positive tubules with internalized transferrin. Time is given as minutes:seconds. This sequence is taken from Movie S3. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "SUPP CAPTION FIGS6.png": "'\\n\\n**Figure S6 \\\\(|\\\\)****GRAF1 BAR+PH overexpression does not affect the uptake of MHC Class I which enter GRAF1-negative compartments.****A and B,** Epifluorescence micrographs of HeLa cells, transiently transfected with GFP-tagged GRAF1 FL (A) or GRAF BAR+PH (B) were pulsed with anti-MHC Class I antibody for 5 (A) or 15 minutes (B) at 37\\\\({}^{\\\\circ}\\\\)C followed by a brief acid wash to remove surface-bound antibody. MHC Class I was visualized using Alexa568-conjugated secondary antibodies. **C,** Quantitation of surface GFP-GPI levels in cells overexpressing this protein with myc-tagged GRAF1 FL or GRAF1 BAR+PH. **D,** Confocal micrographs of HeLa cells treated with siRNA against GRAF1 or a control siRNA and stained for endogenous GRAF1. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "SUPP CAPTION FIGS2.png": "'Figure S.A.: **Supplementary behavioral data in the binding between GAU1 and Dyes1.**\\n\\n'", "SUPP CAPTION FIGS2-1.png": "\"\\n\\n**Figure S2 A-D\\\\(|\\\\) Supplementary biochemical data on the binding between GRAF1 and Dynamin.****A,** Coomassie-stained gel and confirmatory Western blots of coimmunoprecipitation experiments in rat brain cytosol performed with either control pre-immunization serum (pre-serum) or the Ab3 antibody. Bands in the Coomassie-stained gel were identified by mass spectrometry as described. **B and C,** Coomassie-stained gel and Western blots of pull-down experiments from mouse brain lysate (B) or HeLa cell cytosol (C) with beads bound to GST (control) or GST-tagged GRAF1 BAR+PH, or SH3 proteins. The bands in the Coomassie-stained gel were identified by mass spectrometry as described. Note the major band of Dynamin present in the SH3 lanes, which is not present in the control or BAR+PH condition. 'cyt' marks the HeLa cell lysate (positive control) lane. **D,** The upper panel shows a raw trace from isothermal titration calorimetry performed as described. The lower panel shows the fitting of this data to a one-site binding model from which the affinity (shown) can be calculated. GRAF1 SH3 domain and peptide concentrations, as well as injection volumes and times are shown.\\n\\n\"", "SUPP CAPTION FIGS3.png": "'\\n\\n**Figure S3 \\\\(|\\\\) GRAF1-positive endocytic structures are Clathrin-independent and exclude transferrin A-C, Confocal fluorescent micrographs of HeLa cells stained for endogenous GRAF1 and Clathrin (A), transferrin (B) or transferrin receptor (B). The depicted images are used to show some of the different morphologies of GRAF1-positive structures that are observed in these cells. Scale bars = 10\\\\(\\\\mu\\\\)m.**'", "SUPP CAPTION FIGS2-2.png": "'\\n\\n**Figure S2 E \\\\(\\\\mid\\\\) Dynasore inhibits the uptake of dextran and affects the localization of GRAF1. E, Confocal micrographs (maximum projections) of HeLa cells treated with either DMSO (vehicle) or 100\\\\(\\\\mu\\\\)M dynasore for 1 hour before addition of dextran for 15 minutes, fixation, and immunostaining for dextran and the focal adhesion marker paxillin. Scale bars = 10\\\\(\\\\mu\\\\)m.**'"}