Western Blot Analysis of Exosomal Proteins

Cells were lysed in a lysis buffer (Cell Signaling Technology, USA) supplemented with protease inhibitors (Calbiochem, USA) at 4 °C for 30 min, while the exosomes were lysed in 20 μL lysis buffer at 4 °C for 10 min. Total protein concentration was quantified using the BCA protein assay kit (Pierce, USA). Western blotting was performed according to the standard protocol as previously described [12 (link)]. Antibodies used were as follows: phosphorylated-AKT (p-AKT) (1:1000, 4060, Cell Signaling Technology), AKT (1:1000, 4691, Cell Signaling Technology); Cleaved caspase-3 (1:1000, 29,034, Signalway Antibody), Bcl-2 (1:1000, ab196495, Abcam), vascular endothelial growth factor (1:1000, VEGF, ab52917, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, 5174, Cell Signaling Technology), Calnexin (1:1000, 2679, Cell Signaling Technology) and horseradish peroxidase-conjugated secondary antibody (1:5000, Biosharp). The bands were visualized by using enhanced chemiluminescence reagents and analyzed with a gel documentation system (iBrightCL1000, Invitrogen and Image Lab Software version 3.0).