Flotillin 1 clone 18

Samples were electrophoresed on pre-cast 4–12% polyacrylamide bis-tris gels using MOPS running buffer (ThermoFisher, Waltham, MA, USA) and transferred onto 0.2 μM nitrocellulose membranes at 400 mA for 2 h. Thereafter, blots were blocked in Odyssey blocking buffer (Li-COR, Lincoln, NE, USA) and membranes cut to allow the analysis of the same samples for Alix (clone 3A9, 1 μg/mL; Cell Signaling, Danvers, MA, USA), Tsg101 (clone EPR7171(B), 1 μg/mL; Abcam, Cambridge, MA, USA), Flotillin-1 (clone 18, 1 μg/mL; BD Biosciences, San Jose, CA), PrP (clone ICSM-18, 1 μg/mL; D-Gen, London, England), and TOM20 (clone 29, 0.25 µg/mL; BD Biosciences, San Jose, CA, USA). Membranes were incubated overnight at 4 °C in primary antibody solution, followed by 6 × 10 min washes with Tris buffered saline with 0.05% Tween-20 (TBST). Then, membranes were incubated with infrared-labeled goat anti-mouse 800 or donkey-anti-rabbit 680 secondary antibodies (Li-COR, Lincoln, NE, USA) for 1 h at room temperature, followed by 6 × 10 min washes with TBST. Immunoreactive bands were visualized using a Li-COR Odyssey CLx infrared imaging system (Li-COR, Lincoln, NE, USA).

Flow cytometry was performed on a FACS Calibur (BD Biosciences, Breda, The Netherlands) and cell sorting on a FACS Aria (BD) using PE‐labeled monoclonal antibodies against HLA‐A2 (BB7.2; BD Pharmingen, San Diego, CA), HLA‐DR (L243; BD), HLA‐DQ (1a3; Meridian Life Science, Saco, ME), NGFR (C40‐1457; BD Pharmingen) or CD20 (L27; BD). For Western blotting, mouse antibodies against hsc70 (sc‐7298; Santa Cruz Biotechnology, Santa Cruz, CA), gp96 (ab63469; Abcam, Cambridge, UK), 6xHis (ab18184; Abcam), and rabbit antibodies against CD9 (ab65230; Abcam), PTK2B (ab24798; Abcam) and PI4K2B (ab37812; Abcam) were used. As secondary antibodies, biotinylated goat anti‐mouse or anti‐rabbit antibodies (Invitrogen) were used and visualized by Streptavidin‐QDots 625 (Invitrogen) under UV illumination. For detection of EV, mouse antibodies against human CD9 (HI9a, BioLegend, Fell, Germany) and flotillin‐1 (clone 18, BD Biosciences, San Jose, CA, USA) were used.