Exosomal Protein Analysis by Western Blot

Western blotting was performed using the standard SDS-PAGE separation technique as previously reported^{18 (link),19} . The harvested cells were disrupted with RIPA cleavage buffer (Cell Signaling Technology). Proteins were extracted from exosomes using a Total Exosome Protein Isolation Kit (Invitrogen, Carlsbad, CA, USA) following the instructions supplied. The lysates were collected after centrifugation, and the protein concentration was quantified with BCA kit (Thermo Fisher Scientific). 50 mg of protein was added to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with antibodies against phosphor-JNK (ab124956), phospho-MAPK (ab195049), phospho-ERK (ab201015), phospho-NF-κB (ab76302), TRAF-6 (ab33915) and GAPDH (ab8245) purchased from Abcam. Subsequently, the membranes were incubated with a secondary antibody for 1.5 h at room temperature. Immunoreactive protein bands were detected using an Odyssey scanning system (USA), quantified by Image J, and normalized to the corresponding amount of total protein. The experiment was repeated in triplicate with 3 replicates.