Western Blot Analysis of VE-cadherin and Connexin-43

Western blotting was used to detect the expression of VE-cadherin and Connexin-43 on ECs as previously described [26 (link)]. Total proteins from ECs after different treatments were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and then separated with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transfered onto polyvinylidene fluoride membranes (Beyotime, China). Afterwards, membranes were blocked in 3% BSA for 2 hours at room temperature and incubated at 4°C overnight with primary antibodies against VE-cadherin (Abcam) or Connexin-43 (Cell Signaling Technology). The next day, membranes were washed in TBS-T and incubated in HRP-conjugated secondary antibody (Boster biotechnology, Wuhan, China) for 1 hour at room temperature. Then, ECL (Beyotime, China) was applied to detect the bands with a chemiluminescence imaging system (ChemiQ 4800mini; Ouxiang, China).