Western Blot Analysis of EV Proteins

EVs were lysed in a 1:1 dilution with RIPA buffer (Thermo Fisher Scientific) before determining total protein concentration using a bicinchoninic acid protein assay (Thermo Fisher Scientific). Twenty micrograms of total protein was loaded into 4–20% Tris HCl polyacrylamide gels and resolved using the Criterion electrophoresis system (BioRad, Hercules, CA, USA). The resolved proteins were then transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA) using the Criterion transfer system (BioRad). The membranes were blocked in a mixture of 5% non-fat milk and 1× TBS (TBS; BioRad) at room temperature for an hour on an automated rocker. The nitrocellulose membranes were washed twice with 1× TBS and incubated with primary antibodies (annexin A2 (8235; Cell Signaling), GRP94 (20292; Cell Signaling Technology), and HSP70 (ab228421; Abcam)) overnight on an automated rocker at 4 degrees Celsius. Next, the membranes were washed three times with 1× TBS and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (BioRad) at a 1:3000 dilution prepared in blocking solution for an hour on an automated rocker. The nitrocellulose membranes were washed four times with 1× TBS, incubated with ECL reagent (BioRad) for seven minutes, and developed on an imager (BioRad).