Mouse anti tsg101

Loading buffer was added to 10 µg of EV protein sample and boiled at 95 °C for 5 min, separated in a 12% SDS-polyacrylamide gel (SDS-PAGE) and electrophoretic transferred to nitrocellulose membranes. Membranes were blocked in 2.5% nonfat dry milk in 1x TBS-T (0.5% Tween-20), at room temperature and incubated with the following primary antibodies: rabbit anti-CD9 (1:2000) (sc-9148; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-CD10 (1:3000) (GTX111680; Genetex, Irvine, CA, USA), mouse anti-HSC70 (1:2000) (sc-7298; Santa Cruz Biotechnology, Inc.), mouse anti-TSG101 (1:1000) (sc-7964; Santa Cruz Biotechnology, Inc.). Membranes were then washed with 1x TBS-T (0.5% Tween-20). The secondary antibodies used were the mouse IgGκ BP conjugated to HRP (1:2000) (sc-516102; Santa Cruz Biotechnology, Inc.); and the mouse anti-rabbit IgG-HRP (1:2000) (sc-2357; Santa Cruz Biotechnology, Inc.). Secondary antibodies were incubated for 2 h, with agitation at room temperature. Following TBS-T washes, protein bands were detected using the chemiluminescence reagent WesternBright ECL HRP substrate (Advansta Inc., San Jose, CA, USA.) and images acquired with G:BOX Chemi XX9 gel imaging system (Syngene, Cambridge, UK).

To obtain whole-cell extracts, NSPCs were harvested in a RIPA buffer (320 mM sucrose, 50 mM TRIS pH 7.5, 1% Triton X-100, 10% glycerol) and 1% of inhibitor of proteases cocktail (Sigma-Aldrich), incubated on ice for 30 min and centrifuged for 12 min at 13,000 g. Protein amount was quantified by Bradford assay (BioRad, Hercules, CA, USA) and 10–20 μg of protein was subjected to SDS-polyacrylamide gel electrophoresis and transferred overnight onto a polyvinylidene difluoride membrane (Immobilon-PSQ, Millipore). Membranes were blocked in 5% nonfat dry milk and then incubated with the following primary antibodies: rabbit anti-HSP90 (1:300, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-TSG101 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-CD63 (1:1000, BD Biosciences, San Jose, CA, USA), mouse anti-CD81 (1:500, BD Biosciences, San Jose, CA, USA). Blots were washed, incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch, West Grove, PA, USA), then washed again and finally incubated in lumilight-enhanced chemiluminescence substrate (BioRad) and exposed to Chemidoc (BioRad). Densitometric analysis on scanned blots was performed using the ImageLab program (BioRad).

The concentration of exosomal total protein was quantified by the bicinchoninic acid (BCA) assay (Thermo Fisher Inc.) using bovine serum albumin (BSA) as standard. The pellets were also dissolved in 200 μL RIPA buffer for protein assay. The samples were individually homogenized in 5 mM Tris–HCI (4 mM EDTA, pH 7.4, containing 1 M pepstatin, 100 M leupeptin, 100 M phenylmethyl sulfonylfluoride, and 10 g/mL aprotinin) and cleared by centrifugation at 14,000g for 10 min at 4 °C. Approximately 100 μg of protein were run on a discontinuous SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in TBS containing 0.05% Tween 20 and were incubated with the following primary antibodies: (1) Mouse anti-CD9 (sc-13118, Santa Cruz), (2) Mouse anti-CD63 (sc-5275, Santa Cruz), (3) Mouse anti-TSG101 (sc-7964, Santa Cruz), (4) Mouse anti-Alix (sc-53540, Santa Cruz), (5) Mouse anti-calnexin (10,427–2-AP, Promega, Madison), (6) Mouse anti-PD-L1 (sc-293425, Santa Cruz), (7) Rabbit monoclonal anti-E-cadherin (ab194982), (8) Rabbit polyclonal anti-N-cadherin (ab18203) and (9) Mouse anti-GAPDH (sc-47724, Santa Cruz, CA, USA). The optical density (OD) of the signals was quantified and expressed as the ratio of the tested proteins to GAPDH for analysis of protein in cells and to ALIX for analysis of protein in exosomes.