Enhanced chemiluminescence kit

Total protein sample was extracted using RIPA analysis buffer containing protease inhibitor cocktail (Yeasen Biotech, Shanghai, China). 20 µg of protein sample was subjected to SDS-PAGE gel analysis and blotted to the PVDF membrane. After one hour blocking, the membrane was probed using the following primary antibodies overnight at 4°C: CHRDL1 (H00091851-A01, Amylet Scientific, Beijing, China, 1:1000), FAM107A (HPA055888, Amylet Scientific, Beijing, China, 1:1000), actin (abs100041, Absin, Beijing, China, 1:2000). After washing, the membrane was further labeled with HRP conjugated secondary antibody (1:3000; Absin, Beijing, China, abs20040) for 60 min at ambient temperature. Signal development was conducted using an enhanced chemiluminescence kit (Yeasen Biotech, Shanghai, China). The protein bands were photographed using the Gel-Doc gel imager (Bio-Rad, Philadelphia, USA).

Total proteins were extracted using RIPA reagent (Beyotime Biotechnology, Shanghai, China) and the protein concertation was determined by BCA kit (Beyotime Biotechnology, Shanghai, China) as per the instructions. Then, 25 μg of protein was loaded on 10% SDS-PAGE and transferred on PVDF membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk for 1 h, PVDF membranes were incubated with primary antibodies (rabbit anti-RUNX2, 1:1000, ab236639; rabbit anti-OCN, 1:1000, ab133612; rabbit anti-OSX, 1:1000, ab209484; rabbit anti-OPN, 1:1000, ab8448; rabbit anti-GAPDH, 1:1000, ab9485; all purchased from Abcam, Shanghai, China) overnight at 4°C. The following day, membranes were washed 3 times with TBST buffer for 5 minutes each, and further incubated with secondary antibody (goat anti-rabbit H&L preadsorbed, 1:1000, ab7090; purchased from Abcam, Shanghai, China) for 2 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence kit (Yeasen, Shanghai, China) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States). GAPDH functioned as the internal control.

Total protein from veins tissues and SV-SMCs was extracted using RIPA buffer (Beyotime, China). The protein concentrations were determined by BCA assay. Subsequently, equal protein from cell lysates was separated by 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore Corp., Billerica, MA, USA). After blocked with 5% skim milk, the membranes were incubated with the following primary antibodies against SM22α, OPN, FOXC2, Dll4, Notch1, Hey2, and EphrinB2 (all from Abcam) overnight at 4 °C, followed by the horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000; Abcam) at room temperature for 1 h. The protein was detected with an enhanced chemiluminescence kit (YEASEN, Shanghai, China) and the band intensity was quantified with Image J 14.0 software. GAPDH served as the loading control.