Western Blotting for Protein Analysis

Western blotting was performed as previously described^{16 (link)}. For cell culture medium samples, the proteins from 500 µl culture medium was precipitated with methanol and chloroform. The precipitated proteins were re-solubilized with laminal sample buffer and boiled for 5 min before undergoing acrylamide gel electrophoresis. For plasma samples, 20 µg of total protein was used for this analysis. As we lacked house-keeping proteins in the culture medium and plasma, a major protein band from MemCode Reversible Protein Stain Kit (Pierce Biotechnology) for polyvinylidene difluoride membrane was used for loading control. The Western blot bands were corrected for the major protein band after quantification by Image J and expressed as a ratio of western blot band and major protein band.