Xanthine Oxidase-Derived ROS Display a Biphasic Effect on Endothelial Cells Adhesion and FAK Phosphorylation

Figure 5

Effect of orthovanadate, a tyrosine phosphatase inhibitor on ROS-induced dephosphorylation of FAK and ERK1/2. HUVECs were preincubated for 30 min with 100 mM of orthovanadate, washed twice, and then treated with Hx-XO (4.5 mU/mL) for the indicated times. (a) The cells were harvested and the soluble fraction was analysed for FAK activation. FAK was immunoprecipitated using a monoclonal antibody, and its phosphorylation state and expression were analysed by Western blot with anti-phosphotyrosine and anti-FAK antibodies. (b) Cells were harvested and the soluble fraction was analysed by Western blot with anti-phospho-ERK1/2. Total ERK1/2 proteins levels at all times points were equivalent as measured by immunoblot of the same membranes using antibodies against total ERK1/2 coupled to alkaline phosphatase substrate. (c) HUVECs adhesion was determined by loading cells with Calcein-AM as described in Materials and Methods after 60 minutes of Hx-XO treatment. Results are expressed as % of control without Hx-XO (mean ± SEM, . Hx-XO versus untreated controls; ^#orthovanadate versus Hx-XO alone).

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