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. 2019 Dec 31;30:101416. doi: 10.1016/j.redox.2019.101416

Fig. 2.

Fig. 2

Effects of GSTpi on TNF-α-induced Src/VE-cadherin pathway activation. (A, B) The pcDNA3.1 and Flag-GSTpi groups were treated with TNF-α for 30 min, and co-immunoprecipitation of p120 and β-catenin and immunoblotting analysis were performed to assess the binding of VE-cadherin (IP results come from different blots with same samples. *, compared with the pcDNA3.1 group; #, compared with the pcDNA3.1 + TNF-α group). (C, D) The NC-siRNA and GSTpi-siRNA groups were treated with TNF-α for 30 min, and co-immunoprecipitation of p120 and β-catenin and immunoblotting analysis were performed to assess the binding of VE cadherin (IP results come from different blots with same samples. *, compared with the NC-siRNA group; #, compared with the NC-siRNA + TNF-α group). HUVECs were transfected with pcDNA3.1 or Flag-GSTpi and further treated with TNF-α (50 ng/mL). (E) Levels of phospho-p120, phospho-β-catenin, phospho-SHP-2, phospho-Src (Tyr416), and phospho-Src (Tyr527) were measured and calculated (*, compared with the pcDNA3.1 group; #, compared with the pcDNA3.1 + TNF-α group). (F) HUVECs were transfected with NC-siRNA or GSTpi-siRNA and treated with TNF-α (50 ng/mL). Level of phospho-p120, phospho-β-catenin, phospho-Src (Tyr416) and phospho-Src (Tyr527) were measured and calculated (*, compared with the NC-siRNA group; #, compared with the NC-siRNA + TNF-α group). HUVECs were transfected with pcDNA3.1, Flag-GSTpi, or Myc/Src and further treated with TNF-α (50 ng/mL). (G) Levels of phospho-VE-cadherin, phospho-p120, phospho-β-catenin, and phospho-Src (Tyr416) were measured and calculated (TNF-α (50 ng/mL) for 30 min). (H) Cell membrane and cytoplasmic VE-cadherin protein levels in HUVECs were measured and calculated (TNF-α (50 ng/mL) for 1 h). (I) Endothelial cell permeability by detecting the FITC-Dextran 40 fluorescence intensity level and TEER were measured and calculated (TNF-α (50 ng/mL) for 1 h *, compared with the Flag-GSTpi group; #, compared with the Flag-GSTpi + TNF-α group). Data are expressed as means ± SDs (n = 3). Data were analyzed by unpaired Student's t-tests. * or #, P < 0.05; ** or ##, P < 0.01.