Proteins were extracted from different EV populations by RIPA buffer (20 nM Tris-HCl, 150 nM NaCl, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100, pH 7.8) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). The protein content was quantified by BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific). Thirty micrograms of proteins were separated by electrophoresis using a 4–15% gradient sodium dodecyl sulfate–polyacrylamide gel. The proteins were transferred to a PVDF membrane by the iBlot™ Dry Blotting System (Life Technology) and then immunoblotted with the following antibodies: CD63 and CD9 (Santa Cruz Biotechnology) and hepatocyte growth factor (HGF) (Thermo Fisher Scientific). The protein bands were visualized using a ChemiDoc™ XRS+ (BioRad) with an enhanced chemiluminescence detection kit (ECL) (GE Healthcare, Amersham).
To further analyze the protein content of the different EV populations, 100 μg of EV protein was hybridized on glass slides from the Human Cytokine Array kit (Ray Biotech) according to the manufacturer's protocol. The kit allows detecting the expression of 80 different cytokines and growth factors.
To further analyze the protein content of the different EV populations, 100 μg of EV protein was hybridized on glass slides from the Human Cytokine Array kit (Ray Biotech) according to the manufacturer's protocol. The kit allows detecting the expression of 80 different cytokines and growth factors.