Mouse anti alix

EV samples were mixed with sample buffer containing dithiothreitol (DTT), heated to 95 °C for 10 min and separated on 4-12% Bis-Tris polyacrylamide gels (Thermo Scientific). Proteins were electrotransferred to Immobilon-FL polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with 50% v/v Odyssey Blocking Buffer (LI-COR Biosciences) in Tris buffered saline (TBS). All immunolabeling was performed with 50% v/v Odyssey Blocking Buffer in TBS containing 0.1% Tween 20 (TBS-T). Primary antibodies were used overnight at 4 °C and included rabbit anti-CD9 antibody (Abcam, clone EPR2949, 1:2500 dilution), rabbit anti-TSG101 (Abcam, ab30871, 1:1000 dilution), mouse anti-Alix (Abcam, clone 3A9, 1:1000 dilution), rabbit-anti-EGFR (Cell Signaling Technology, clone D38B1, 1:1000 dilution) mouse-anti-β-actin (Cell Signaling Technology, clone 8H10D10, 1:1000 dilution), and mouse-anti-Myc (9E10 from MYC 1-9E10.2 hybridoma, ATCC, 1:4000). Secondary antibodies included Alexa Fluor 680-conjugated anti-rabbit antibodies (Thermo Fisher Scientific, A-21,076, 1:7500 dilution) or IRDye 800CW anti-mouse antibodies (LI-COR Biosciences, 926-32212, 1:7500 dilution). Imaging was performed on an Odyssey Infrared Imager (LI-COR Biosciences, Leusden, The Netherlands) at 700 and 800 nm, respectively.