Overexpression of p120 results in hypophosphorylation of VE-cadherin, which in turn, regulates TEM. (A) HUVEC monolayers were infected with a low or a high dose of p120GFP (6 μL, 17.5 μL in Figure 2A) or GFP (2 μL in Figure 2C), lysed, and subjected to Western blot for phospho–VE-cad–Tyr658, total VE-cad, and β-actin. A representative blot is shown from 4 experiments performed. The graph represents normalized values obtained by densitometry analysis, indicating the relative absorbance of phospho–VE-cadherin–658 with respect to total VE-cadherin for each condition and normalized with the GFP values, and are the mean plus or minus SD of 4 different experiments. β-actin is shown as a loading control. (B) HUVECs were transduced with GFP or a high dose of p120GFP, treated with TNF-α, and incubated with control IgG beads or anti–ICAM-1 beads for 10 minutes and lysed in hot sample buffer. Samples were then diluted to allow immunoprecipitation with 4G10 mAb, and blotted for phospho–VE-cad–Tyr658 or phospho–VE-cad–Tyr731. A representative blot is shown from 3 independent experiments. The graph represents normalized values obtained by densitometry analysis corresponding to phospho–VE-cad divided by the total VE-cad input, and with respect to the GFP-IgG beads value, and is the mean plus or minus SD of 3 separate experiments. *P < .05; **P < .01. (C,D) HUVECs were infected with p120GFP, GFP, VE-cad, or the mutated version of VE-cad at Tyr658 or Tyr731. Overexpression of VE-cad Y658F and Y731F strongly inhibited TEM (C; **P < .01; *P < .05 with respect to WT), whereas total accumulation of PMNs was similar for every condition tested (D). Values represent the mean plus or minus SD from 3 different experiments.