Pvdf membrane

The cell lysis buffer (Cell Signaling Technology, California, USA) containing protease and phosphatase inhibitor cocktail (Abcam, USA) was used for the extraction of total proteins from the chondrocytes, followed by quantification with a BCA kit (Shanghai Ze Ye Biotechnology Co., Ltd, Shanghai, China). After loading approximately 30 μg protein, 12% SDS PAGE was used to separate the proteins, which were further transferred to the PVDF membrane (Cell Signaling Technology, California, USA). Then, the protein-loaded PVDF membrane was mixed with 5% skim milk to block the nonspecific binding proteins, followed by incubation in the solution of primary antibody against SOX-9 (1:1000, R&D, Minnesota, USA), Nrf2 (1:2000, R&D, Minnesota, USA), DPP-4 (1:2500, R&D, Minnesota, USA), Lamin B1 (1:500, R&D, Minnesota, USA) and GAPDH (1:800, R&D, Minnesota, USA). The membrane was subsequently incubated with the secondary antibody (R&D, Minnesota, USA). Lastly, the bands were visualized using the enhanced chemiluminescence (ECL) kits (Cell Signaling Technology, California, USA) on a Tanon-2500 imaging system (Shanghai, China), followed by quantifying the relative expression level of target proteins with Image J software ²⁵.

Preparation of protein extracts from subconfluent cell cultures or resected tumors using RIPA buffer as well as western blot analysis was performed as described elsewhere [58 (link)]. In brief, 80 µg of proteins were separated in a 8.5% v/v sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Fisher Scientific, Schwerte, Germany) using a semi-dry transfer system (Bio-Rad, Munich, Germany). After blocking with 5% w/v skimmed milk for at least 1 h, PVDF membrane was incubated with goat anti-human EphB4 IgG (R&D, AF3038; 0.2 µg/mL) or rabbit anti EphrinB2 IgG (Novus Biologicals, Littleton, CO, USA, NBP1-48551; 3.1 µg/mL) for 2 h at room temperature and afterwards overnight at 4 °C. Membranes were washed three times and incubated with the appropriate horseradish peroxidase coupled secondary antibodies (rabbit anti-goat IgG, Sigma Aldrich, Taufkirchen, Germany; A5420; goat anti rabbit IgG, Sigma Aldrich, A0545) for 1 h at room temperature. Protein bands were detected with Super Signal West Pico, Dura, or Femto Chemiluminescent Substrate (Fisher Scientific) and the MF-ChemiBis 3.2 imaging system (Biostep, Burkhardtsdorf, Germany). To verify equal protein loading, membranes were stripped and reprobed with mouse anti β-actin IgG (Sigma Aldrich, A5316) and horseradish peroxidase coupled rabbit anti-mouse IgG (Sigma Aldrich, A9044).

Total proteins were extracted from the cells or exosomes by using a lysis buffer (Cell Signaling Technology, California, USA) and quantified with a BCA kit (Shanghai Ze Ye Biotechnology Co., Ltd., Shanghai, China). Approximately 30 μg of proteins was loaded, separated through 12% SDS PAGE, and transferred to a PVDF membrane (Cell Signaling Technology, California, USA). Then, the protein-loaded PVDF membrane was mixed with 5% skim milk to block nonspecific binding proteins and incubated with a solution of primary antibodies against PD-L1 (1:800, R&D, Minnesota, USA), PD-L2 (1:800, R&D, Minnesota, USA), GRP 94 (1:800, R&D, Minnesota, USA), HSP70 (1:800, R&D, Minnesota, USA), HSP90 (1:800, R&D, Minnesota, USA), CD9 (1:800, R&D, Minnesota, USA), CD81 (1:800, R&D, Minnesota, USA), PD-1 (1:800, R&D, Minnesota, USA), and GADPH (1:800, R&D, Minnesota, USA) [19 ]. The membrane was subsequently incubated with the secondary antibody (R&D, Minnesota, USA). Lastly, bands were visualized with ECL kits (Cell Signaling Technology, California, USA), and the relative expression levels of target proteins were quantified with Image J software.