Quick star bradford protein assay

F4ac fimbriae were isolated and purified according to a previously published method [10 (link)], the only modification being the introduction of filtration of solubilized F4ac fimbriae through 0.20 μm pore size filters (Nylon Membrane 0.2 μm 47 mm PK/100, Millipore Corporation, MA, USA) before each step of precipitation at pH 4 during the purification process to prevent aggregation [5 (link)]. The concentration of isolated fimbriae was determined with Quick Star Bradford protein assay (Bio-Rad Laboratories, Mississauga, Ont., Canada), the purity by Sodium Dodecyl Sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) separation and Coomassie staining and the identity by Western Blot technique using rabbit anti-F4ac polyclonal antibodies (Dr J.M. Fairbrother, Faculte de Medecine Veterinaire, Universite de Montreal, Saint-Hyacinthe, QB, Canada) as previously described [5 (link)]. The isolated F4ac fimbriae protein was stored at −20 °C in PBS.

Protein concentration was measured in EV samples using the Bradford method (Quick Star Bradford Protein Assay, Bio-Rad, Hercules, CA, USA, USA). EV markers were characterized by Western blotting. Each EV sample was mixed with 2× Laemmli sample buffer (Bio-rad, USA). The resulting EV samples were applied to the wells of a 10% PAAG gel in a volume of 10 μL (50 μg of protein). The proteins were separated at a field strength of 80 V in the concentrating gel and 180 V in the separating gel. This was followed by the semi-dry electrotransfer of proteins from the gel to a PVDF membrane using a Trans-Blot Turbo Transfer system (Bio-rad, USA). Following blocking, the membrane was incubated with the primary antibodies (Table S1) anti-C;D81 (Affinity Biosciences, DF8045, 1:1000), anti-CD63 (Affinity Biosciences, AF5117, 1:1000), anti-CD9 (Affinity Biosciences, AF5139, 1:1000) for 18 h at +4 °C overnight and then, after washing, with secondary antibodies—for 1 h at room temperature. The membrane was developed with an ECL substrate with signal detection in an automated ChemiScope instrument (Clinx, Shanghai, China).

Cell surface proteins of hCMEC/D3 were biotinylated according to the manufacturer's instructions (Pierce Cell Surface Protein Isolation Kit; Pierce Biotechnology, Rockford, IL, USA). Briefly, hCMEC/D3 were washed twice with ice-cold PBS and labeled with sulfo-NHS-SS-biotin at 4°C for 30 min. Cells were collected, washed with TBS and lysed in IP Lysis Buffer (Pierce™ Direct Magnetic IP/Co-IP Kit; Pierce Biotechnology, Rockford, IL, USA) with protease inhibitors (Halt™ Protease Inhibitor Single-Use Cocktail; Pierce Biotechnology, Rockford, IL, USA) by incubating on ice for 30 min with gentle vortexing every 10 min. The cell lysate was centrifuged at 13,000 rpm for 20 min. The supernatant was collected and protein concentration was measured by bicinchoninic acid (BCA) protein assay (Pierce BCA Protein Assay Kit; Pierce Biotechnology, Rockford, IL, USA). The biotinylated cell surface proteins were isolated via magnetic beads (Pierce™ Direct Magnetic IP/Co-IP Kit; Pierce Biotechnology, Rockford, IL, USA) coupled to anti-biotin antibody (Rabbit Anti-Biotin Antibody ab53494; Abcam Inc., Cambridge, MA, USA). The proteins eluted from the magnetic beads were quantified with Bradford protein assay (Quick Star™ Bradford Protein Assay; Bio-Rad Laboratories Inc., Hercules, CA, USA).