Western Blots SDS-PAGE and Western blot analysis were performed as described [10] using ECL detection (Amersham, Buckingham- shire, UK). Levels of the proteins of interest were estimated by image scanning of non-saturated ECL exposures and corrected by the band intensities of the Coomassie staining of membranes or by the signal given by an antibody against a non-related protein (RhoGDI for most experiments). Fluorescence Microscopy Cells were cultured on glass bottom dishes (Mattek Corp., Ashland, MA), transfected with fluorescent constructs, treated with the indicated agents and visualized directly on a confocal microscope (LEICA DMRE2, Heidelberg, Germany). For IF cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% SDS, and incubated with primary antibodies and secondary Alexa-Fluor conjugated antibodies (Invitrogen), counterstained with DAPI (Invitrogen) and mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). Unless otherwise stated, images shown are single Z-section images for co- localization assessment. All experiments were repeated at least three times and representative results are shown. Electron Microscopy EM analysis was carried out at the EM Facility of Centro de Biolog´ıa Molecular Severo Ochoa (CSIC, Madrid). For conven- tional EM analysis cell monolayers were fixed in situ with 2% glutaraldehyde in So¨rensen phosphate Na/K buffer (pH 7.4). Fixed cells were processed for embedding in Epoxy, TAAB 812 Resin (TAAB Laboratories, Berkshire, England). Subsequently, cells were subjected to postfixation with 1% osmium tetroxide/ 0.8% potassium ferrocyanide, incubation with 0.15% tanic acid, incubation with 2% uranyl acetate and dehydration in increasing concentrations of ethanol. Infiltration of the resin was accom- plished by increasing concentrations of Epon/ethanol. After polymerization of infiltrated samples, ultrathin sections were stained with saturated uranyl acetate and lead citrate and examined at 80 kV in a Jeol JEM-1010 (Tokyo, Japan) electron microscope. For immuno-electron microscopy cells were processed by freeze substitution. Cells were fixed in situ with 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). Cells were embedded in 10% gelatine, cryopro- tected with 30% glycerol and frozen in liquid nitrogen. Freeze substitution was carried out at 285uC in methanol containing 0.5% uranyl acetate in an Automatic Freeze-Substitution system (AFS, Leica). Samples were later infiltrated with Lowicryl HM20 and polymerized by UV irradiation at 245uC for two days. Ultra thin Lowicryl sections were obtained in a Reichert-Jung Ultracut E ultramicrotome. Samples were examined as above. For immunogold labelling the ultra thin sections were placed sequentially on top of drops of the different solutions: TBS (30 mM Tris-HCl, pH 8.0, 150 mM NaCl), 0.2 M ammonium chloride in PBS, blocking buffer (TBS +0.1% BSA and 1% gelatine) and rabbit anti-GFP antibody (Invitrogen) diluted 1:100 in blocking buffer. After washing, grids were incubated with protein A-15 nm gold conjugate (Cell Microscopy Center. University Medical Center. Utrecht, The Netherlands). After washing, grids were air dried and counterstained with 2% aqueous uranyl acetate and 1% lead citrate. Grids were examined as above. Estimation of Protein Stability Protein half-life was evaluated by time-course experiments in the presence of CHX and Western blot analysis. Stability of the Lysosomal Degradation Sequence various constructs used was assessed by cell transfection followed by tripsinization to split cells in identical aliquots. After attachment to substrate, cells were treated with CHX and construct levels were estimated by Western blot. Quantification and Statistics The degree of colocalization of the different constructs was analyzed with software from Leica and Scion Corporation for image analysis. Results are presented as average values 6 SD or SEM, as indicated. Average values were compared by Student’s t- test for unpaired observations. Supporting Information Figure S1 Co-localization of RhoB and lysosomal proteases. (a) The distribution of RhoB (red) and cathepsin B (CB, green) in BAEC treated with vehicle or 10 mM chloroquine for 24 h was assessed by IF, nuclei were stained with DAPI (blue); bar, 20 mm. Graphs represent the fluorescence intensity profile along the lines depicted in the images, showing the points of co-localization. Results are representative of three assays. (b) The percentage of cells showing five or more points of co-localization of RhoB and cathepsin B was calculated by monitoring 40 cells per experimen- tal condition from three different experiments and it is shown as average values 6 standard error of mean (SEM). Found at: doi:10.1371/journal.pone.0008117.s001 (6.04 MB TIF) Figure S2 Effect of autophagy modulation on RhoB levels and degradation. (a) MEFs from wt and Atg5(2/2) mice were incubated with CHX to block new protein synthesis and the levels of endogenous RhoB were assessed at the indicated times by Western blot. (b) BAEC were treated in the absence or presence of 10 mM 3-methyladenine (3-MA) for 24 h and RhoB levels were estimated as above. Dotted lines show sites where lanes from the same gel have been cropped. Results are average values 6 SEM of three experiments. (c) BAEC were pretreated with 3-MA for 30 min, after which, CHX was added and levels of RhoB were analyzed at the indicated times. Results are average values 6 SEM of three experiments. Found at: doi:10.1371/journal.pone.0008117.s002 (2.29 MB TIF) Figure S3 Effect of lysosomal inhibitors on the size and pattern of RhoB-positive vesicles. (a) BAEC were transfected with GFP- RhoB as in Fig. 2c and the size distribution of RhoB-positive vesicles was determined by measuring 60 randomly taken RhoB- positive vesicles per experimental condition from three experi- ments. The proportion of vesicles of the indicated sizes is expressed as percentage of the total. (b) Multivesicular pattern of chloro- quine-elicited GFP-RhoB and Lamp1-GFP vesicles in BAEC transfected as in Fig. 2c. Bars, 5 mm. Found at: doi:10.1371/journal.pone.0008117.s003 (1.92 MB TIF) Figure S4 Effect of U18666A on RhoB stability. BAEC were treated with 10 mM U18666A for 24 h. Levels of endogenous RhoB and of RhoGDI (as control) were assessed by Western blot at the indicated times after CHX addition. Results shown are average values 6 SEM of four experiments. Found at: doi:10.1371/journal.pone.0008117.s004 (1.76 MB TIF) Figure S5 Co-localization of GFP-RhoB with cholesterol in control or NPC fibroblasts. Control or NPC fibroblasts were transfected with GFP-RhoB. Co-localization with cholesterol was evaluated in fixed cells stained with filipin. Bars, 20 mm. Co- localization is shown as average values 6 SEM of determinations from three experiments (*p,0.05 vs control). Found at: doi:10.1371/journal.pone.0008117.s005 (3.21 MB TIF) PLoS ONE | www.plosone.org 12 December 2009 | Volume 4 | Issue 12 | e8117