Chemiluminescence gel imaging system

Prepared protein samples (10 μg) were subject to 12% SDS-PAGE, followed by electro-transfer onto PVDF membranes. Then the membranes were blocked in 5% skimmed milk for 1 h. Antibodies against exosome markers CD9 (ab92726; Abcam, Cambridge, UK), CD81 (ab109201; Abcam) and calnexin (ab133615; Abcam) were diluted at a ratio of 1:1000. The inflammatory response of vascular tissue and VSMCs was detected using antibodies against NFATc3 (18222-1-AP, 1:1000 dilution; Proteintech, Wuhan, China) and IL-6 (DF6087, 1:500 dilution; Affinity Biosciences, Cincinnati, USA), IL-1β (DF6251, 1:500 dilution; Affinity Biosciences) and TNF-α (ab205587, 1:500 dilution; Abcam). The internal control anti-β-actin primary antibody (TA-09; Zhongshan Jinqiao Biotechnology, Beijing, China) was used at 1:1000 dilution. The PVDF membranes were incubated with the above primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated goat anti-mouse IgG (ZB-2305, 1:4000; Zhongshan Jinqiao Biotechnology) or HRP-conjugated goat anti-rabbit IgG (ZB-2301, 1:4000; Zhongshan Jinqiao Biotechnology) secondary antibodies for 1 h at room temperature. After extensive wash, protein bands were visualized using ultra high sensitivity ECL kit (HY-K1005; MCE, New Jersey, USA), and images were obtained with a chemiluminescence gel imaging system (12003153; Bio-Rad, Hercules, USA).

The Human Phospho‐MAPK Array Kit (ARY003B, R&D Systems®, Inc. USA) was performed to detect the relative phosphorylation levels of 43 human kinases. Chemiluminescence Gel Imaging System (Bio‐Rad, USA) was used to detect the array's chemiluminescence signal (details of the steps are in Method S1).

After 7 days of cell culture in each group, the cells were lysed by Radio Immunoprecipitation Assay to extract total proteins, and the protein concentration was estimated by Enhanced BCA Protein Assay Kit (Beyotime, China). The same amount of sample protein was added to a 10% SDS-PAGE well and transferred to a hydrophobic Polyvinylidene Fluoride (PVDF) membrane. After blocking the membrane antibody with 8% skim milk for 60 min, it was immersed in the primary antibody solution of Runx2, BMP2 and OPN (Beyotime, China) at a ratio of 1:1000 and overnight at 4°C. The membrane was washed three times with quickblock™ blocking buffer (Beyotime, China), and then incubated with secondary antibody solution of HPR-labeled goat anti-rabbit IgG (Beyotime, China) at a ratio of 1:1000. The PVDF membrane was washed with quickblock™ blocking buffer and then reacted with ECL developing reagent. The bands were exposed by chemiluminescence gel imaging system (Bio-Rad, USA).