Ecl chemiluminescence staining assay kit

The pellets of sEVs and lEVs were lysed with RIPA buffer (Pierce, Rockford, IL, USA) on ice for 40 min. Insoluble material was pelleted by centrifugation for 15 min at 11,000 × g at 4°C. Supernatants were transferred to a new tube, and the protein concentrations were quantified with a BCA Protein Assay kit (Beyotime, Shanghai, China) according to the manufacture’s protocols. Next, 20 μg of protein was loaded into gels for separation by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) after which the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk powder, primary antibodies were added, and the membrane was incubated at 4°C overnight. Primary antibodies used were CD9 (C4, Santa Cruz Biotechnology, Dallas, TX, USA; 1:500), CD81 (B11, Santa Cruz; 1:1000), CD63 (H5C6, BD Biosciences, Franklin Lakes, NJ, USA; 1:1000), Tsg101 (4A10, Abcam, Cambridge, UK; 1:2000), GM130 (4A10, Abcam, Cambridge, UK; 1:500) and Calnexin (Roteintech Group, INC; 1:1000). After washing with PBS, the membrane was further incubated with the secondary antibody for 1 h at room temperature. Proteins were visualized using an ECL chemiluminescence staining assay kit (Bio-Rad) and the density of each protein band was quantified.