Golm1

Exosomal protein was extracted using a cocktail of radio-immunoprecipitation assay (RIPA) buffer (Sangon Biotech Co., Ltd., China) and the protein inhibitor, phenylmethylsulfonyl fluoride (PMSF) solution (Thermo Fisher Scientific, Waltham, MA, USA). Bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China) was performed to measure the protein concentration and 10 μg of the protein separated by 10% SDS-PAGE gel electrophoresis. The separated proteins were then bloated onto polyvinylidene fluoride (PVDF) membranes (Merck KGaA), and blocked in 5% skimmed milk for 2 h at room temperature. The membrane was then incubated overnight with the following primary antibodies: CD63 (Cat: 67605-1-Ig, dilution: 1: 7000, Proteintech, China), CD9 (Cat: 60232-1-Ig, Dilution 1: 10,000, Proteintech, China), GOLM1 (Cat: HPA010638, dilution: 1: 5000, Sigma-Aldrich, Beijing, China), and β-actin (Cat: A5441, dilution: 1: 5000, Sigma-Aldrich, Beijing, China), all diluted as instructed by the manufacturer. They were then incubated in corresponding HRP secondary antibodies (Goat anti-mouse: Cat: 12-489, dilution: 1: 6000, goat anti-rabbit: Cat: 12-348, dilution: 1: 6000, all from Sigma-Aldrich, Beijing, China) and the bands on the PVDF membrane detected using the Chemiluminescent Substrate System (Thermo Fisher Scientific, USA).