Anti cd81 b 11

For each group (CTR, BPH, PCa) 4 mL of plasma was pooled and Size Exclusion Chromatography (SEC) was performed for the isolation of plasma-derived exosomes, as described previously [27 (link)]. Exosomes from plasma of CTR, BPH and PCa patients were lysed in CHAPS buffer 1 × containing Tris 10 mM pH 7.4, MgCl2 1 mM, ethyleneglycoltetraacetic acid (EGTA) 1 mM, CHAPS 0.5%, glycerol 10%, phenylmethylsulfonyl fluoride (PMSF) 1 mM and protease inhibitor cocktail (1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 µg/mL aprotinin, and PMSF 1 mM).
Protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Thirty micrograms of exosomal lysates were resolved on 10% acrylamide gel and transferred to a Protran BA85 nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). Nonspecific binding sites were blocked by incubation in PBS containing 0.05% Tween 20 and 5% milk powder. Blotting was performed employing anti-Tsg101 (4A10, GeneTex, Irvine, CA, USA), and anti-CD81 (B-11, Santa Cruz Biotechnology, Dallas, TX, USA) monoclonal antibodies, for 18 h at 4 °C. After incubation with appropriate anti-mouse peroxidase-conjugated secondary antibody (IgG; Amersham Biosciences, Milan, Italy) for 1 h at room temperature, membranes were revealed by enhanced chemiluminescent (ECL) substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Small extracellular vesicles harvested for protein extraction were isolated as previously described, however following the final ultracentrifugation, the pellet was lysed immediately in RIPA buffer supplemented with protease inhibitors on ice. To facilitate further lysis, samples were probe sonicated on ice. Protein concentration was determined using the Bio-Rad Protein Assay. 10μg of protein was loaded per well. Samples were resuspended in Laemmli buffer, resolved using the NuPAGE® Bis-Tris Electrophoresis System with NuPAGE 10% Bis-Tris Protein Gels and transferred onto PDVF membranes. Antibodies used included: Anti TSG101 EPR7130(b) (Abcam; 1:1000), Anti CD63 (Abcam; 1:1000), Anti CD81 B-11 (Santa Cruz; 1:500), Anti ALIX 3A9 (Cell Signaling; 1:1000), Anti Flotillin-1 18 (Biosciences; 1:1000), HRP- anti rabbit secondary (GE Healthcare; 1:5000) and HRP- anti mouse secondary (GE Healthcare; 1:5000). Western blots were developed on X-ray film using a SRX-101A table top film processor.

To perform Western blot analysis, the plasmatic exosomes pellet was resuspended in 1 ml of PBS and it was further purified by using 30% sucrose in deuterium oxide (D₂O, ACROS Organics, fisher scientific, USA) density gradient ultracentrifugation for 18 h at 110,000 g, in order to eliminate contaminants⁴⁶ (link)^,47 . Density gradient ultracentrifugation was performed by using TH-641 Rotor (ThermoFisher Scientific, USA). The 12 fractions obtained were washed in PBS for 1 h at 110,000 g and then were resuspended in CHAPS buffer 1x for subsequent experimental analysis.
Lysates were prepared in CHAPS buffer (10 mM Tris-HCl [pH 7.4], MgCl₂ 1 mM, EGTA 1 mM, CHAPS 0.5%, glycerol 10%, β-mercaptoethanol 5 mM, PMSF 1 mM) containing protease inhibitor cocktail. Exosomes lysates were subjected to electrophoresis on SDS polyacrylamide gels and transferred to nitrocellulose membranes (ProtranWhatman, Dassel, Germany). After blocking in 5% dry milk in PBS 1X, membranes were hybridised with primary antibodies: M75⁴⁸ (link), anti-CD81 (B-11, Santa Cruz Biotechnology, USA), anti-Alix (3A9, ThermoFisher Scientific, Waltham, MA, USA) mouse monoclonal antibodies. After incubation with appropriate peroxidase-conjugated anti-IgG (AmershamBiosciences, Milan, Italy), membranes were revealed using the ECL Chemiluminescent Substrate, (ThermoFisher Scientific, USA).