Western Blot Analysis of Exosomal Markers

Radio-immunoprecipitation assay buffer (SenBeiJia Biological Technology Co., Ltd., Nanjing, China) was applied to collect total protein from tissues and cells. The protein concentration was measured bicinchoninic acid protein assay kit (Beyotime, China). About 40 μg of protein was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, electro-blotted onto PVDF membrane (Millipore, USA) and blocked with 5% skimmed milk. Antibodies were showed as follows: anti-CXCL17 (AF4207, 1:1000) from R&D Systems (MN, USA), CD63 (sc-5275, 1:1000) and TSG101 (sc-7964, 1:1000) from Santa Cruz Biotechnology (CA, USA), Alix (2171, 1:1000) from Cell Signaling Technology (MA, USA), and horseradish peroxidase-conjugated secondary antibody (1:10,000). The protein expression was detected by Quantity-one software (Bio-Rad Laboratories, USA) with enhanced chemiluminescence kit [26 (link)].