Mechanisms for VCAM-1 activation of ERK1/2 in HMVEC-L.

<p>Treatment of HMVEC-L cells overnight with 10ng/ml TNF-α induced VCAM-1 expression (data not shown). <b>A</b>) Confluent monolayers of TNF-α-treated HMVEC-Ls were nontreated (NT) or treated with 27 µg/ml anti-VCAM-1 plus 15 µg/ml of a secondary antibody to crosslink and stimulate VCAM-1. Phosphorylation of ERK1/2 Thr202/Tyr204 (P-ERK1/2) and total expression of ERK1/2 was examined by western blot using rabbit anti-phospho ERK1/2 Thr202/Tyr204 (1/1000) followed by HRP-conjugated anti-rabbit (1/2000) and ECL detection. <b>B</b>) Confluent monolayers of TNF-α stimulated HMVEC-L cells in 12 well plates were nontreated or incubated for 30 minutes with the solvent control DMSO, apocynin (4 mM), Gö-6976 (2.3 nM) or CinnGEL 2-methylester (10 µM). These endothelial cells were then stimulated with anti-VCAM-1 antibody plus a secondary antibody for 15 minutes. The apocynin, DMSO, Gö-6976 or CinnGEL 2-methylester had no effect on endothelial cell viability as determined by trypan blue exclusion and had no effect on VCAM-1 expression as determined by flow cytometry (data not shown). Representative western blots are shown. Data presented are the mean ± standard deviation from 3 experiments. The phosphorylation status of ERK1/2 is presented as the fold increase in the ratio of the relative intensity of P-ERK1/2 divided by the relative intensity of the loading control (total ERK1/2). *, p<0.05 compared to <b>A</b>) NT cells or <b>B</b>) anti-VCAM-1 stimulated cells.