β-arrestin1 C-terminus tail induces loss of barrier integrity and VE-cadherin down-regulation. (A) VE-cadherin surface expression was tested by flow cytometry in HUVEC that stably expressed mock (pink) or CFP-myc-tagged β-arrestin1 full-length (1–418, green), ΔC (1–374, orange) and C-tail (375–418, blue). Isotype control (Ig) is shown in grey. (B) Mock and C-tail HUVEC were subjected to VE-cadherin internalization assay (iVEC) and analyzed by confocal microscopy. Graph represents the mean + s.e.m. of the percentage of cells with iVEC staining; n >300; T-test: *** p < 0.001. (C) Three day-old mock and C-tail HUVEC were stained for VE-cadherin (VEC), β-catenin (β-cat) and p120-catenin (p120) and analyzed by confocal microscopy. Scale bar: 10 μm. (D) Relative permeability was measured in mock, FL, ΔC and C-tail HUVEC by the fluorescence of 40 kDa FITC-dextran passage. Graph shows the mean ± s.e.m. of 3 independent experiments. Two-way ANOVA test: *** p < 0.001. (E-F) Protein and RNA expression of indicated targets were tested using western-blot (E) and RT-PCR (F) in mock and C-tail HUVEC. Cad5: cadherin-5, VE-cadherin; Cad2: cadherin-2, N-cadherin. Densitometry analysis was done with Image J software. T-test: * p < 0.05. (G-H) HUVEC were transfected with luciferase reporter for VE-cadherin promoter activity and Renilla. (G) They also received a control CFP-myc plasmid (mock) and the CFP-myc-tagged FL, ΔC and C-tail β-arrestin1. Alternatively, they received pGFP (mock) and GFP-tagged β-arrestin2 comprising amino acids 1–410 (FL), 1–359 (ΔC) and 317–410 (C-tail). (H) Two days prior plasmid transfection, cells were treated with non-silencing duplexes (nsi) and siRNA targeting βarr1, βarr2, or both (βarr1/2). Graphs show the mean ± s.e.m. of 3 independent experiments. Two-way and one-way ANOVA test: ** p < 0.01; * p < 0.05. All panels are representative of at least 3 independent experiments.