Immunoblotting

Ultracentrifuged EV were re-suspended in lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1%Igepal, 10 % glycerol, 10 mM MgCl2, 1 mM EDTA, 25 mM NaF, 1 mM Na2VO4, plus proteinase inhibitors), and incubated 20 min on ice. Non-soluble material was eliminated by centrifugation. Protein concentrations were determined using the Bradford assay. Samples were then subjected to SDS-PAGE and the proteins were transferred onto Polyvinylidene fluoride (PVDF) membranes from GE Healthcare (Barcelona, Spain), as previously described [51]. Non-specific binding was blocked with 5 % non-fat dry milk. Primary antibodies were rabbit α-CD63 and α-CD81 (Exo AB Antibody Kit – System Biosciences) and rabbit α-CD73 (ab124725, Abcam Inc.). Incubation with the primary antibodies at the appropriate dilution was performed overnight at 4 °C. As secondary antibody an anti-rabbit IgG horseradish peroxidase-conjugated from Amersham Biosciences was used. Incubation with the secondary antibody was performed at room temperature for 1 h. Blots were visualized using chemiluminescence using ECL-Plus reagent (Amersham Biosciences).