Exosomal Protein Profiling Methodology

Protein samples were extracted from MSC-exosomes, cells or tissues according to a previously described protocol (Wang et al., 2009 (link)). Equal amounts of protein were subjected to 10% SDS-PAGE and immobilized on PVDF membranes. The membranes were blocked in skimmed milk for 1 h at room temperature and then incubated with different primary antibodies overnight. The source and dilutions of antibodies are as follows: rabbit anti-CD63 (1:500 dilution; Abcam, UK), rabbit anti-CD9 (1:400 dilution; Abcam), rabbit anti-CD81 (1:400 dilution; Abcam), rabbit anti-cytochrome c (1:1,000 dilution; Abcam), rabbit anti-TSG101 (1:400 dilution; Abcam), rabbit anti-p-STAT3 (1:50,000 dilution; Abcam), rabbit anti-STAT3 (1:1,000 dilution; Abcam), and anti-Sema3A (1:1,000 dilution; Abcam). The antibody against GAPDH (1:1,000 dilution; Sigma-Aldrich) was used as an internal control. After incubation with secondary antibodies (goat anti-rabbit antibody, 1:5,000 dilution; Biosharp, USA), the proteins on membranes were visualized by incubation with a chemiluminescent reagent (Millipore Corporation, USA) and exposure on Kodak films.