Western Blot Analysis of Cellular Proteins

Western blot analysis was performed as previously described 52 (link), 53 (link), 54 (link).Protein lysates/extracts (40 μg) were prepared from the BMMSCs, ECs, and exosomes using RIPA lysis buffer (Thermo Fisher Scientific) and loaded on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) independently. Separated proteins in the gels were transferred to polyvinylidene difluoride (PVD) membranes using an electrotransfer apparatus (Bio-Rad, USA). Following blocking the membrane with 5 % non-fat dry milk (1 h), the membranes were probed with a primary antibody (anti-RUNX2 (ab23981), anti-Bglap (ab13420), anti-Tie-2 (ab111074) and anti-phosphor Tie2 (Y992, ab151704), anti-NOS3 (ab76198) and anti-phosphor NOS3 (S1177, ab230158), anti-CD9 (ab223052), anti-CD63 (ab8219)) for 2 h at 4 °C. Then membranes were further incubated with a secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were developed with the ECL Western blotting detection system (GE Healthcare, Piscataway, NJ, USA) and the image was recorded using a gel documentation system (Bio-Rad, USA). Each target protein band density was normalized with a respective GAPDH density using Image Lab densitometry software (Bio-Rad) and expressed as fold changes to control.