Extracellular Vesicle Protein Isolation and Characterization

Protein isolation and Western blot were carried out in the same manner as previously described [46 (link),47 (link),48 (link)]. Proteins from EVs were extracted with RIPA buffer (10 mM Tris, 150 nM NaCl, 1% deoxycholic acid, 1% Triton X, 0.1% SDS, and 1 mM EDTA) containing protease inhibitors (aprotinin, PMSF, and sodium orthovanadate). A Pierce^TM bicinchoninic acid (BCA) protein assay kit was used to assess protein content (Thermo Fisher Scientific, Waltham, MA, USA). A 4–20% Tris-Glycine gel (Bio-Rad, Hercules, CA, USA) was loaded with equal quantities of protein (20 g/lane) and electrophoresed under nonreducing conditions. Proteins were transferred to a PVDF membrane, blocked for 2 h in blocking buffer (PBS, 0.05 percent Tween20^®, 5% milk), and then probed overnight with the following primary antibodies: ALIX (Sc-166952; Santa Cruz, CA, USA); CD81 (Sc-7637; Santa Cruz); and GM130 (#12480; Cell Signaling, Danvers, MA, USA). The membranes were rinsed the next day and treated for 1 h at room temperature (RT) with the secondary antibodies donkey anti-mouse IRDye 800CW and donkey anti-rabbit IRDye 680RD (1:2000, LI-COR Biosciences, Lincoln, NE, USA). All antibodies were diluted according to the manufacturer’s instructions. A fluorescence scanner was used to photograph the membranes (Odyssey v.3.0, LI-COR Biosciences).