*4.13. Western Blotting*

Cells in the exponential differentiation phase were diluted to a cell suspension of <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> cell/mL using complete medium. Cells were seeded into a 6-well plate at 2 mL/well in an incubator at 37 ◦C and 5% CO2. When the cells spread across 70% of the bottom of the 6-well plate, the complete medium was replaced with serum-free medium with or without DGS drug for 24 h.

HUVECs cells were washed and scraped out after centrifugation at 4 ◦C, followed by washing in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Triton, 0.5% NP40, 1 nM PMSF). After centrifugation at 10,000 rpm for 30 min, the supernatant was collected. The protein concentrations were determined using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Each lane was loaded with 20 µg of the protein and separated. The proteins were then loaded on each lane and separated on SDS-PAGE gels, followed by electrophoretic transfer to nitrocellulose (NC) membranes (MilliPore, Boston, MA, USA). The membranes were blocked with 5% BSA for 2 h and then incubated with primary antibodies at 4 ◦C. The primary antibodies were diluted in the BSA buffer, and secondary antibodies were diluted in TBST. The antibody-reactive bands were revealed by chemiluminescence. The images were scanned and band intensities were analyzed with Image J software.
