Effect of antioxidants and dominant-negative p67phox on tyrosine phosphorylation of VE-cadherin and α-catenin. (A) HUVEC monolayers were exposed to TNF in the presence or absence of NAC or MnTBAP. Lysates were subjected to immunoprecipitation using anti–VE-cadherin antibody. Half the immunoprecipitates were probed with antiphosphotyrosine antibody (top blot), and the other half were probed with anti–VE-cadherin antibody to confirm adequate immunoprecipitation (bottom blot). (A) TNF caused robust tyrosine phosphorylation of VE-cadherin, whereas both NAC and MnTBAP prevented such phosphorylation. (B) HUVECs were infected with Ad-lacZ (control), the active p47phox mutant, Ad-p47SD, or Ad-DNp67 before TNF exposure. Ad-DNp67 prevented TNF-induced tyrosine phosphorylation of VE-cadherin and α-catenin (top panel). Adp47SD caused mild increases in basal VE-cadherin and α-catenin phosphorylation. Histogram (bottom) represents densitometry values for VE-cadherin phosphorylation.