Anti proteases cocktail

Cell pellets were lysed in standard RIPA buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with anti-proteases cocktail (Roche, Milan, Italy) and anti-phosphatase cocktails 2 and 3 (Merck Millipore). Protein concentration was determined by Bradford assay (Pierce, Milan, Italy). Fifty µg of total proteins were prepared in 1× Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% (w/v) glycerol, 4% β-Mercaptoethanol, 0.05% bromophenol blue), denatured for 5 min by boiling, and separated by SDS-PAGE. Resolved proteins were transferred to nitrocellulose membrane (Santa Cruz Biotechnology, Heidelberg, Germany) and, after blocking with 5% non-fat dry milk, the filters were incubated with anti-HA (Merck Millipore) (1:2000) or anti-actin (BioLegend, London, UK) as a loading control overnight (o/n) at room temperature (RT). After being washed three times with PBS-T (phosphate buffered saline, 0.05% Tween-20), membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (Merck Millipore) for 1 h at RT. Immunocomplexes were revealed using the TMB-Blotting 1-Step Solution (Pierce). Reported figures were representative of at least three independent experiments.

For protein extraction, EV-pellets (obtained after ultracentrifugation of a pool of four plasma samples, total 6 ml) were resuspended in 100 μl lysis buffer containing 300 mM NaCl, 50 mM Tris pH 7.4, 0.5% NP-40 and anti-proteases cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and 15 μg total protein was submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ponceau staining was performed before blocking with 5% milk. Primary commercially available antibodies used were CD63 1:1,000 (CBL553, Millipore, Billerica, MA, United States), RAB27B 1:250 (HPA019849, Sigma-Aldrich), Flotillin 1:500 (ab41927, Abcam, Cambridge, MA, USA), HSP70 1:1,000 (EXOAB-Hsp70A-1, System Biosciences, Mountain View, CA, USA), TSG101 1:500 (ab4A10, Abcam, Cambridge, MA, USA) and RAB7A 1:1,000 (ab50533, Abcam, Cambridge, MA, USA).