Ib401002

Small EV (5 µg total protein) were separated by sodiumdodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–20% gradient gel (4561094, Bio-Rad, Hercules, CA, USA), transferred to PVDF membranes (IB401002, Thermo Fisher Scientific Inc.), and blocked with 5% non-fat dry milk dissolved in Tris-buffered saline containing 0.05% Tween 20 (Merck KGaA; TBST). The following proteins were analysed: CD63 (1:500, PA5-92370, Thermo Fisher Scientific Inc.), CD81 (1:1000, PA5-79003, Thermo Fisher Scientific Inc.), CD73 (1:1000, ab175396, Abcam, Cambridge, United Kingdom), Calnexin (1:1000, ab22595, Abcam) and GM130 (1:1000, MA5-35107, Thermo Fisher Scientific Inc.). Horseradish peroxidase-coupled donkey anti-rabbit antibody (NA9340, Merck KGaA) was used as secondary antibody in a 1:1000 dilution. The binding of the antibodies was detected using the chemiluminescent Amersham ECL Prime Western Blotting Detection Reagent (GEHERPN2232, Cytiva, Marlborough, MA, USA) on a C-DiGit Blot Scanner (LI-COR Biosciences, Lincoln, NE, USA).

The total protein concentration was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific Europe bv) following the manufacturer’s instructions. Western blotting was performed using a 4–12% Bis-Tris protein gel, with MES SDS running buffer and transfer onto a polyvinylidene difluoride membrane (respectively NP0322Box, NP0002, and IB4010-02, Thermo Fisher Scientific Europe bv). For Western blotting, a 10 µg/sample was used for lysates and a fixed amount of supernatant. Proteins were visualized using antibodies against Core (Dako, Glostrup, Denmark) and Pol (Santa Cruz Biotechnology, Dallas, TX, USA); goat anti-rabbit IgG horseradish peroxidase (GE Healthcare Life Sciences, Hoegaarden, Belgium) and goat anti-mouse IgG (H + L) horseradish peroxidase (Thermo Fisher Scientific Europe bv) secondary detection systems were employed. Read out was performed using SuperSignal West Femto substrate (Thermo Fisher Scientific Europe bv).