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. 2012 Nov 1;23(21):4212–4225. doi: 10.1091/mbc.E12-01-0038

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© 2012 Timmerman et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Thrombin induces a transient drop in the TER of endothelial monolayers and transiently increases tyrosine phosphorylation of VE-cadherin–associated β-catenin. (A) HUVECs were cultured to confluency on FN-coated electrode arrays. At time point 0, cells were incubated with (dashed line) or without (solid line) thrombin and electrical resistance was monitored in time by ECIS. Thrombin rapidly decreased the resistance of the endothelial monolayer, which was restored after 2–3 h. (B) HUVECs were cultured on FN-coated glass covers, grown to confluency, and stimulated with thrombin for 5 or 30 min, as indicated. Immunostaining for VE-cadherin (green) and phosphotyrosine (pY, red) shows colocalization at cell–cell junctions after 5 min of thrombin stimulation (merged image, colocalization appears in yellow). Bar, 20 μm. (C) VE-cadherin was immunoprecipitated from control or thrombin-treated HUVECs, and phosphorylation levels were determined by Western blotting (WB) with an anti-phosphotyrosine antibody. Top, thrombin induces an increase in tyrosine phosphorylation (pY) of a ∼92-kDa cadherin-associating protein, likely β-catenin. Bottom, equal levels of VE-cadherin and catenins were immunoprecipitated. Top bar graph, fold increase in tyrosine phosphorylation relative to the levels of VE-cadherin–associating β-catenin. Bottom bar graph, relative binding of β-catenin to VE-cadherin during thrombin stimulation. Results are means ± SD of four independent experiments. (D) β-Catenin was immunoprecipitated from control or thrombin-treated HUVEC, and phosphorylation levels were determined by Western blotting. (E) HUVECs were stimulated with thrombin and lysed, and IP for VE-cadherin was performed (1st IP). The washed pellet was incubated with lysis buffer containing 1% SDS, and the supernatant was recovered and subjected to a second IP using pY antibodies to precipitate tyrosine-phosphorylated proteins from the dissociated VE-cadherin complex (2nd IP; pY). Blot shows increased tyrosine phosphorylation of VE-cadherin–bound β-catenin after 5 min of thrombin treatment, which was reduced after 30 min.