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Figure 2 | Cell Communication and Signaling

Figure 2

From: The C-terminus region of β-arrestin1 modulates VE-cadherin expression and endothelial cell permeability

Figure 2

β-arrestin1 C-terminus tail binds to S665D VE-cadherin mutant. (A) HUVEC were transfected with CFP-myc-tagged β-arrestin1 (CFP-βarr1, green). After serum deprivation, cells were subjected to VE-cadherin internalization assay, stimulated with VEGF (50 ng/ml, 15 min) and fixed for staining (iVEC, red). Cells were analyzed by confocal microscopy. Scale bar: 10 μm. (B-D) After overnight serum starvation, three day-old HUVEC were stimulated with VEGF (50 ng/ml) for the indicated times. (B-C) The anti-VE-cadherin (VEC) and anti-β-arrestin1/2 (βarr1/2) immunoprecipitated (IP) fractions were analyzed by western-blot. Densitometry analysis was done with Image J software. T-test: * p < 0.05. (D) Total cell lysates (TCL) were analyzed by western-blot for S665 phosphorylation of VE-cadherin (pVEC) and total VEC. (E) HEK-293T cells were co-transfected with CFP-myc-tagged β-arrestin1 (myc-βarr1) together with control plasmid (−), wild-type (WT), non-phosphorylable (SV) or phosphomimetic (SD) VE-cadherin mutants. TCL and IP fractions were analyzed by western-blot as indicated. (F) HEK-293T cells were transfected with CFP-myc-β-arrestin1 (myc-βarr1, +) or CFP-myc control plasmid (−). Cellular extract was incubated with recombinant histidine-fused VE-cadherin intracellular domain (ICD), either WT or SD. TCL and pull-down fractions were analyzed by western-blot as indicated. (G) Schematic representation of human β-arrestin1, with two arrestin (arr) domains. The C-terminus tail (375–418) comprises an AP2 binding site (LIE and RXR in blue) and two phosphorylation sites (in red). (H-J) HEK-293T cells were co-transfected with VEC SD mutant together with control plasmid (−), full-length and deleted forms of myc-βarr1. IP fractions and TCL were analyzed by western-blot. (J) HEK-293T cells were co-transfected with VEC SD mutant together with a constant amount of full-length myc-βarr1 (**) and increasing concentrations of CFP-myc-tagged β-arrestin1 C-terminus tail comprising amino acids 475–418 (myc-C-tail, *). TCL and IP fractions were analyzed by western-blot. All panels are representative of at least 3 independent experiments.

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