1. Volume 39, Issue 3, Pages 468-476 (August 2010)
S-Nitrosylation of β-Catenin by eNOS-Derived NO Promotes VEGF-Induced Endothelial Cell Permeability 
Sébastien Thibeault, Yohann Rautureau, Malika Oubaha, Denis Faubert, Brian C. Wilkes, Chantal Delisle, Jean-Philippe Gratton 
Molecular Cell 
Volume 39, Issue 3, Pages (August 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2010 39, 468-476DOI: (10.1016/j.molcel.2010.07.013)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 NO Donors Induce S-Nitrosylation of β-Catenin
(A) Representative tracing of triiodide-dependent chemiluminescence detection of S-nitrosylation (SNO) of β-catenin immunoprecipitates. β-catenin (injection marks 3 and 4) or control immunoprecipitates (IgG; injection marks 5 and 6) from BAEC lysates were treated with GSNO (1 mM; marks 4 and 6) or vehicle (marks 3 and 5) and washed extensively. Immunoprecipitates were injected into the triiodide reaction vessel, and SNO levels were recorded. SNO levels were compared to amounts released by injections of GSNO standards (1 and 2.5 pmol; marks 2 and 1, respectively). Equal immunoprecipitations levels, prior to injection in the reaction vessel, were confirmed by western blotting (10% of IP; right inset). See also Figure S1.
(B) BAEC lysates were immunoprecipitated using anti-β-catenin (top) or anti-VE-cadherin (bottom) antibodies (IP). Immunoprecipitates were treated with SNAP and subjected to biotin-switch assays. SNO levels of proteins present in immunoprecipitates were detected by western blot (wb).
(C) BAEC were treated with SNAP for 15 min. Whole-cell lysates were subjected to biotin-switch assays, and SNO was detected by anti-β-catenin, anti-VE-cadherin, or anti-α-catenin western blotting. Data are representative of three separate experiments.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 VEGF Induces eNOS-Dependent S-Nitrosylation of β-Catenin
(A) BAEC were stimulated with VEGF (40 ng/ml), and SNO levels of β-catenin were determined using the biotin-switch assay followed by western blot (wb). The means ± SEM of four experiments are presented. ∗p < See also Figure S2.
(B) VEGF induces S-nitrosylation at intercellular contacts. BAEC were stimulated with VEGF (40 ng/ml) or SNAP (1 mM) for 15 min. Fixed cells were subjected to a biotin-switch assay. S-nitrosylation was visualized using Alexa-488-conjugated avidin (green). β-catenin immunostaining was performed in parallel (red). Nonspecific staining was determined in control cells incubated with Alexa-488 avidin. Arrowheads indicate the presence of S-nitrosylation at intercellular junctions (merge with β-catenin).
(C) BAEC were treated with the NOS inhibitor L-NMMA (0.1 mM) prior to stimulation with VEGF (40 ng/ml; 15 min). Cell extracts were processed for biotin-switch assays, and SNO of β-catenin was determined. Data are the means ± SEM of five separate experiments. ∗p < 0.05.
(D) Mouse lung endothelial cells isolated from WT or eNOS−/− mice were treated with VEGF (40 ng/ml; 15 min). Cell extracts were processed for biotin-switch assays, and SNO of β-catenin was determined.
(E) Wild-type and eNOS−/− mice were injected intravenously with VEGF (2 μg; i.v.) or saline. Extracts from whole lungs were processed for biotin-switch assays, and SNO of β-catenin was determined. Total levels of eNOS and β-catenin in lung extracts are presented.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Cystein 619 Is a Major Substrate of S-Nitrosylation
(A) Representative nESI-MS/MS spectrum obtained from S-nitrosylated β-catenin. GST-β-catenin was incubated with GSNO, and tryptic peptides containing S-nitrosylated Cys619 were identified. The peptide sequence is shown above (y ions in red, b ions in blue). Y series of ions containing the S-nitrosylated cysteine were found to have a neutral loss of 29 Da (yx − NO).
(B) Mapping of S-nitrosylation sites. COS-7 cells were transfected with Myc-tagged β-catenin mutants, and whole lysates from transfected cells were treated with CysNO for 15 min. SNO levels of β-catenin mutants were determined by biotin-switch assays and anti-Myc western blotting.
(C) β-catenin is S-nitrosylated at Cys619 by endogenously produced NO. Myc-tagged WT or C619S-β-catenin was cotransfected with S1179S-eNOS in COS-7 cells, and SNO of β-catenin was determined by biotin-switch. Data are the means ± SEM of five separate experiments. ∗p < 0.05.
(D) VEGF-stimulated (40 ng/ml; 15 min) S-nitrosylation of β-catenin at Cys619. BAEC were transfected with Myc-tagged WT or C619S-β-catenin. SNO in response to VEGF (40 ng/ml; 15 min) was determined by biotin-switch assays and anti-Myc western blotting.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
S-Nitrosylation of β-Catenin at Cys619 Promotes Dissociation from VE-Cadherin and Endothelial Permeability
(A) Three-dimensional representation of β-catenin nitrosylated at C619. The H3 helix of Arm11 (magenta) contains Cys619, and the H3 helix of Arm12 (cyan) contains Tyr654.
(B) Dissociation of β-catenin from VE-cadherin by eNOS-derived NO is dependent on Cys619. Lysates from COS-7 cells transfected with Myc-tagged WT or C619S-β-catenin, VE-cadherin, and S1179D-eNOS were subjected to anti-Myc immunoprecipitates (IP). The presence of VE-cadherin in the anti-Myc immunoprecipitates was determined by western blotting elutes. Data are the means ± SEM of six separate experiments. ∗p < 0.05.
(C) VEGF-stimulated dissociation of β-catenin from VE-cadherin is inhibited by C619S-β-catenin. BAEC were transfected with Myc-tagged WT or C619S-β-catenin and stimulated with VEGF (15 min). Cell lysates were subjected to anti-β-catenin immunoprecipitations (IP), and presence of VE-cadherin in anti-β-catenin immunoprecipitates was determined by western blotting elutes. Relative ratios are the means of four separate experiments. SEM were all less than 10%. ∗p < 0.05.
(D) S-nitrosylation of Cys619 does not affect Src-mediated tyrosine phosphorylation of β-catenin. Cell lysates from COS-7 cells transfected with Myc-tagged WT or C619S-β-catenin and cotransfected with c-Src and/or eNOS expression plasmids were immunoprecipitated with anti-Myc antibodies. Tyrosine phosphorylation levels of β-catenin were detected by western blotting elutes with anti-phospho-tyrosine and anti-phospho-Tyr654-β-catenin antibodies.
(E) Inhibition of VEGF-stimulated endothelial permeability by C619S-β-catenin. BAEC monolayers transfected with GFP or with Myc-tagged WT, C350S-, C466S-, or C619S-β-catenin were stimulated with VEGF (40 ng/ml; 30 min). The permeability of endothelial monolayers was measured by the diffusion of FITC-labeled dextran. Data are means ± SEM from three separate experiments performed in triplicates. ∗p < See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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