Gel image system

Cells, exosomes, and synovial tissues were lysed with RIPA lysis buffer (Thermo, USA) to extract total proteins. An equal amount of 35 μg proteins were loaded and divided by SDS-PAGE, blotted to PVDF membranes. The blots were blocked in 5% non-fat milk, followed by incubation with primary antibodies against CD63, CD81, TSG101, GPR94, HDAC4, p-IκBα, p52, RELB, and GAPDH overnight at 4°C. Next day, the proteins were incubated with HRP-conjugated secondary anti-mouse or anti-rabbit antibodies, and visualized by using ECL reagent (Sigma) in a gel image system (BD Bioscience). All antibodies were purchased from Abcam (USA) and used following manufacturer’s description.

Cells derived from wound area were collected and lysed by chilled RIPA buffer containing protease and phosphatase inhibitors (Sigma). Total proteins were quantified by BCA kit (Thermo, USA), loaded, and separated by SDS-PAGE gels, transferred to NC membranes, incubated with anti-Notch1, anti-Dll4, anti-Jagged1, anti-Hes1 and anti-βactin antibodies overnight at 4 °C. Next day, the proteins were hatched with corresponding secondary anti-rabbit or anti-mouse antibody (1:5000, Abcam), and visualized by the enhanced chemiluminescence (ECL) reagents (Thermo) in a gel image system (BD Biosciences, NJ, USA). The gray values of the blots were quantified using Image J software and presented as bar charts. All antibodies were purchased from Abcam and diluted at ratio of 1:2000 in accordance with the manufacturer's protocols.