Anti tsg101 4a10

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. Cell lysates and exosomes were subjected to electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Protran Whatman, Dassel, Germany). After blocking in 5% dry milk in PBS1X, membranes were hybridised with primary antibodies: goat anti-mouse IgG HRP (Amersham Biosciences, Milan, Italy), anti-Tsg101(4A10, GeneTex, Irvine, CA, USA) and anti-GAPDH (GA1R, Santa Cruz Biotechnology, Dallas, TX, USA) monoclonal antibodies. After incubation with appropriate peroxidase-conjugated anti-IgG (Amersham Biosciences, Milan, Italy), membranes were revealed using the ECL Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA, USA).

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).

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. Cell lysates and exosomes were subjected to electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Protran Whatman, Dassel, Germany). After blocking in 5% dry milk in PBS1X, membranes were hybridised with primary antibodies: M75⁴⁷ (link), anti-Tsg101 (4A10, GeneTex, USA), anti-GAPDH (GA1R, Santa Cruz Biotechnology, USA) and anti-actin (C4, Santa Cruz Biotechnology, USA) monoclonal antibodies. After incubation with appropriate peroxidase-conjugated anti-IgG (Amersham Biosciences, Milan, Italy), membranes were revealed using the ECL Chemiluminescent Substrate, (ThermoFisher Scientific).