Protran ba85 nitrocellulose membrane

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).

Briefly, subconfluent cells were lysed in CHAPS buffer 1× (Tris 10 mM pH 7.4, MgCl₂ 1 mM, ethyleneglycoltetraacetic acid (EGTA) 1 mM, CHAPS 0.5%, glycerol 10%, and phenylmethylsulfonyl fluoride (PMSF) 1 mM) with protease (1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 µg/mL aprotinin, and PMSF 1 mM) incubated for 30 min on ice and centrifuged for 30 min at 12,000 rpm at 4 °C, thus removing cell debris and collecting the supernatant. Exosomes were lysed in CHAPS buffer 1× and processed as previously described. Protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Thirty micrograms per sample were resolved on 10% acrylamide gel and transferred to a Protran BA85 nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). Membranes were blocked overnight with 5% dry milk in PBS 1×. Blotting was performed employing anti-Alix (3A9, ThermoFisher Scientific, Waltham, MA, USA), anti-Tsg101 (4A10, GeneTex, Irvine, CA, USA), and anti-CD81 (B-11, Santa Cruz Biotechnology, Dallas, TX, USA) monoclonal antibodies. After incubation with appropriate peroxidase-conjugated anti-immunoglobulin G (IgG; Amersham Biosciences, Milan, Italy), membranes were revealed by enhanced chemiluminescence (Pierce, Rockford, IL, USA).

Western Blot analysis was performed as described in Bischof et al. (2017). Cells were broken by vortexing in the presence of glass beads (diameter 0.25–0.5 mm). Equal protein amounts were loaded on a 15% SDS‐PAGE gel. Proteins were blotted on a protran BA85 nitrocellulose membrane (Schleicher & Schuell, Germany) with 250 mA for 90 min at RT. The membrane was blocked with MTBS‐T (25 mm Tris pH 7.6, 137 mm NaCl, 0.1% Tween 20, 5% milk powder) for 90 min at RT and washed for 30 min with TBS‐T. The primary anti‐GFP‐antibody (SC‐9996; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted in MTBS‐T (1:1000) and incubated overnight at 4°C under constant shaking. The blot was washed three times with TBS‐T (3 × 10 min at RT) and incubated for 2 h at RT with a horse anti‐mouse IgG antibody [no. 7076; NEB/Cell Signaling (Ipswich, USA)]. The secondary antibody was diluted in MTBS‐T 1:3000. Detection was carried out with the Pierce (Thermo Fisher Scientific, Waltham, MA, USA) ECL western blotting substrate according to the manufacturer's instructions.