5 μg per well) in serum-free media for 1–6 h at 37°C or 4°C. When indicated, AlexaFluor-555 transferrin

(25 μg/ml) or AlexaFluor-555 selleck screening library cholera toxin B subunit (10 μg/ml) were added Smoothened Agonist ic50 to cells five minutes prior to the addition of vesicles. For inhibition experiments, cells were pretreated with inhibitors (methyl-β-cyclodextrin, 10 mM; methyl-α-cyclodextrin, 10 mM; sucrose, 0.45 M; chlorpromazine, 1 μg/ml; filipin, 5 μg/ml; cytochalasin D, 1 μg/ml; NiCl2, 2 mM) for 30 min, and the inhibitors remained in the media during incubation with vesicles. All subsequent steps were carried out on ice and ice-cold Dulbecco’s phosphate-buffered saline (PBS) was used for washes. Following incubation with vesicles, cells were washed twice to remove unbound vesicles. Cell exteriors were labeled in one of two ways, as indicated in figure legends: 1) Cells were incubated with AF633-conjugated wheat germ agglutinin (WGA; 25 min, on ice) and washed twice, or 2) Cells were incubated with 6-((biotinoyl)amino)hexanoic acid, succinimidyl ester (Biotin-X, SE; 10 min, on ice), washed twice, and then incubated with AF633-conjugated streptavidin (15 min, on ice) and washed twice. Cells were then fixed in 2% paraformaldehyde, mounted with ProLong AntiFade reagent, and visualized on a Nikon Eclipse TE200. Immunofluorescence Clathrin and caveolin immunofluorescence was performed essentially

as described [14]. Following incubation with vesicles, monolayers Lonafarnib were washed, cell exteriors were labeled with Biotin-X, SE/AF633-Streptavidin and fixed as described above. Fixed cells were washed, permeabilized (0.1% Triton X-100 in Hanks 7-Cl-O-Nec1 solubility dmso buffer; 15 min, 25°C), blocked (5% goat serum and 0.1% bovine serum albumin in permeabilization buffer; 20 min, 25°C), incubated with mouse anti-caveolin-1 or anti-clathrin antibodies (BD Biosciences; 2.5 μg/ml in permeabilization buffer; 1 h, 25°C), washed, and then labeled with AF555-conjugated goat anti-mouse secondary

antibody (μg/ml in permeabilization buffer; 30 min, 25°C), and washed. Following incubation with secondary antibodies, slides were mounted and visualized as described above. For TRAPα and tubulin immunofluorescence, fixed monolayers were permeabilized in PBS supplemented with 1 mM DTT, 1 mM PMSF, and 0.015% digitonin (to release cytoplasmic contents) for 5 min. Permeabilized cells were blocked with 1% BSA in PBS (30 min, on ice), incubated with rabbit anti-TRAPα or mouse anti-β-tubulin primary antibodies (2 μg/ml, in blocking buffer, 1 h, on ice), washed, and incubated with AF555-conjugated goat anti-mouse or anti-rabbit secondary antibodies (30 min, on ice). Following incubation with secondary antibody, slides were mounted and visualized as described above. Leucine aminopeptidase assay Assays were performed using the substrate Leu-p-nitroanilide (0.6 mM in 50 mM Tris-HCl, 1 mM CaCl2, pH 8.3) as described previously [44]. Samples were preincubated with 0.