G box xt4 imager

Immunoblot analyses were performed as previously described with minor modifications [33 (link)]. Briefly, exosomal total protein was harvested using RIPA buffer including 1x Halt™ Protease- and Phosphatase-Inhibitor-Cocktail (ThermoFisher Scientific, Waltham, MA, USA). Using Bradford quantification, 25 µg of protein was denaturized and separated by a 4–12% gradient Bis-Tris polyacrylamide gel electrophoresis (Invitrogen^TM, ThermoFisher Scientific). The proteins were then transferred onto a nitrocellulose blotting membrane (Amersham^TM Protran^TM, Amershan, UK) using a wet blotting approach. After blocking for 1 hour at RT, the blots were incubated with following primary antibodies: anti-CD9 (ERP2949) (Abcam), anti-CD63 (H-193) (Santa Cruz), anti-CD81 (NBP2-20564) (Novus Biologicals, Centennial, CO, USA), anti-TSG101 (4A10) (Abcam), anti-GPC-3 (AF2119, R&D Systems) and anti-calreticulin (Cell Signaling, Danvers, MA, USA; Cat. Nr. 2891) overnight at 4 °C on an orbital shaker. The next day, the blots were washed with 1 x PBS/0.05% Tween for at least 30 min, followed by the incubation with horseradish peroxidase (HRP) coupled secondary antibodies (Cell Signaling, anti-mouse Cat. Nr. 7076, anti-rabbit Cat. Nr. 7074) for 1 hour at RT. The blots were washed again and developed with Immobilon Western chemiluminescent HRP substrate using a G:Box XT4 imager (Syngene, Cambridge, UK).