Mechanisms for VCAM-1 activation of ERK1/2 in endothelial cell lines.

<p>Monolayers of mHEVa cells were nontreated (NT) or incubated for 30 minutes with apocynin (4 mM), PD98059 (30uM), U0126 (40 µM), Gö-6976 (2.3 nM), CinnGEL 2-methylester (10 µM), or catalase (5000 U/ml) where indicated. These are the optimal doses for these inhibitors as we have previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Matheny1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Deem1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-AbdalaValencia1" target="_blank">[8]</a>. At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). After treatement with the inhibitor, the endothelial cells were stimulated with anti-VCAM-1 antibody plus a secondary antibody to crosslink VCAM-1 for 30 min under static conditions. We examined phosphorylation of <b>A,B,F</b>) ERK1/2 Thr202/Tyr204 (P-ERK1/2), <b>C</b>) PKCα Thr638 (P-PKCα), or <b>D</b>) MEK1/2 Ser217/221 (P-MEK1/2) by western blot. <b>E</b>) mHEVa cells were treated with exogenous 1 µM H₂O₂ for 10–60 minutes and ERK1/2 Ser217/221 phosphorylation was determined by western blot. The phosphorylation status of ERK1/2 Thr202/Tyr204, PKCα Thr638, or MEK1/2 Ser217/221 is presented as the fold increase in the ratio of the relative intensity of the phosphorylated enzyme to total ERK1/2, total PKCα or total MEK1/2 expression. Representative western blots are shown and data are presented as the mean ± standard deviation from 3 experiments. (<b>A</b>, <b>B</b>) *, p<0.05 less than anti-VCAM-1-treated group. (<b>C–F</b>) *, p<0.05 greater than NT.