Anti cd63

The RIPA lysis buffer (Solarbio, Beijing, China) was used to conduct the purification of the protein of the cells and hUCMSCs-EVs. The concentrations of extracted protein were tested and calculated by a bicinchoninic acid protein quantification kit (Thermo Scientific, 23225). A 40 μg of denatured protein was subjected to 10% SDS polyacrylamide gel, subsequently transferred onto the polyvinylidene fluoride membranes (Millipore Corp, Billerica, United States). And then these polyvinylidene fluoride membranes were incubated with first antibodies, anti-IL-4 (Boster, 10K274), anti-TNF-α (Proteintech, 17590-1-AP), anti-MMP13 (Proteintech, 18165-1-AP), anti-CD63 (Bioss, bs-1523R), anti-TSG101 (Abclonal, A1692), anti-CD81 (Bioss, bs-6934R), anti-CALNXIN (Proteintech, 10427-2-AP), or anti-GAPDH (Sangon Biotech Co., Ltd, China) at 4 °C for 12 h after blocking with 5% milk. The secondary antibody, anti-rabbit IgG (Sangon Biotech Co., Ltd, Shanghai, China), was used at 37 °C for one hour. The expression level of each protein was assessed by the Odyssey CLx imaging systems (Li-COR Biosciences, Lincoln, United States).

Exosome preparations, normalized to 20 μg protein content, were mixed with 5× Laemmli sample buffer (Elpisbio, Daejeon, Korea) and boiled for 5 min at 95 °C. Exosome proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4%–20% gradient polyacrylamide gels (Cat# 456–1093; Bio-Rad, CA, USA) and subsequently transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk solution in 1× Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) for 1 h, the membrane was incubated overnight at 4 °C with anti-CD63 (1:1000, bs-1523; Bioss, MA, USA), anti-Alix (1:1000, ab88388; Abcam, Cambridge, UK), anti-CD81 (1:1000, bs-6943R; Bioss, MA, USA) and anti-Annexin V (1:1000, SC-8300; Santa Cruz Biotechnology, CA, USA) primary antibodies. The membrane was washed 5 times in 1× TBST for 1 h at room temperature (RT) and then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (1:2000 in TBST containing 5% skim milk; Cell Signaling Technology, MA, USA). After washing five times in 1× TBST for 1 h at room temperature, immunoreactive bands were detected using the Clarity Western Enhanced Chemiluminescence (ECL) kit (Bio-Rad, Cat #: 170–500) and Chemidoc imaging system (Bio-Rad).