Immunoblot Analysis of Cellular and Extracellular Proteins

Cell homogenates were prepared as described by Gong et al. [30 (link)]. Immunoblots were performed essentially as described in our previous studies [31 (link)]. The protein concentrations of homogenates were determined using the method of Bradford [32 (link)] (Bio-Rad, Richmond, CA, USA). Aliquots of cell homogenates containing 100 ng–50 µg of protein or 1–30 µL of extracellular vesicles were separated by SDS-PAGE on gels containing 8% acrylamide (for VE-cadherin), 14% (for CD63 and CD81) or 10% (for all other protein targets). Polyacrylamide gels were run under reducing conditions or non-reducing conditions (for CD63, CD81 and VE-cadherin). Proteins were blotted onto Immobilon-P membranes (SIGMA-Aldrich). ProSieve Protein Colored Markers standards (Lonza Walkersville, Inc.) were used to calibrate the gels. For most targets, immunoblots were developed using enhanced chemiluminescence reagents (ECL Prime, GE Healthcare Biosciences, Pittsburgh, PA, USA); Alix and CD81 blots were reacted with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific Inc.). Chemiluminescence was detected by exposing blots to X-ray film.