Exosome Isolation and Exosome Protein Extraction

The cell medium was centrifuged at 500 g for five minutes and at 2000 g for thirty minutes at 4 °C to remove cellular debris and large apoptotic bodies. After centrifugation, media was added to an equal volume of a 2× polyethylene glycol (PEG, MW 6000, Sigma, 81,260) solution (final concentration, 8%). The samples were mixed thoroughly by inversion and incubated at 4°C overnight. Before the tubes were tapped occasionally and drained for five minutes to remove excess PEG, the samples were further centrifuged at maximum speed (15,000 rpm) for 1 h at 4°C. The resulting pellets were further purified using 5% PEG and then stored in 50–100 μl of particle-free PBS (pH 7.4) at − 80°C. The average yield was approximately 300 μg of exosomal protein from 5 ml of supernatant. Exosome was re-suspended in 150ul RIPA buffer (Beyotime, P0013B) and incubated on ice for 1h. The lysate was centrifuged for 20min at 16000g at 4°C, and the supernatant was further concentrated by ultracentrifuge filter (Millipore, 3kDa cutoff). Protein concentration was measured by BCA assay (Beyotime, P0012).