r84 Inhibits VEGFR2 Figure 2. r84 reduces endothelial cell migration and signaling in vitro. A, A modified Boyden chamber migration assay was used to assess the effect of r84, bevacizumab (bev) on VEGF-induced endothelial cell (EC) migration. 20,000 HDMEC, PAE-KDR, PAE-Flt-1 cells were plated on 8.0 mm cell culture inserts and allowed to migrate overnight towards SFM or human VEGF (100 ng/mL) +/2 500-fold molar excess antibody (bev, r84, control IgG). r84, bev block the VEGF-induced migration of VEGFR2-expressing ECs (A, HDMEC, PAE-KDR). Bev blocks VEGF-induced migration of endothelial cells expressing VEGR1, but r84 does not (A, PAE-Flt-1). Western blots of VEGF-induced signaling in PAE-KDR (B), HDMEC (C), and PAE-Flt-1 (D) lysates following stimulation of cells with 50 ng/mL human VEGF +/2 500-fold molar excess antibody (bev, r84, control IgG). r84 and bev block p-VEGFR2 and downstream phosphorylation (p38, PLCc, ERK1/2) (B, C), but only bev blocks VEGF-induced VEGFR1 phosphorylation in PAE-Flt-1 stimulated cells (D). *p,0.05, **p,0.01, ***p,0.001, statistical differences in A compared to VEGF alone, unless otherwise indicated. doi:10.1371/journal.pone.0012031.g002 towards VEGF (Figure 2A, PAE-Flt-1). To further evaluate the specificity of r84 to mouse VEGF, migration assays were performed with PAE-KDR cells using mouse VEGF as the chemotactic agent. As seen with human VEGF, mouse VEGF significantly induced the migration of PAE-KDR cells as compared to SFM alone (Figure S1A). Only r84 was able to significantly inhibit this migration, while bevacizumab and a control IgG had no effect on cell migration (p,0.001, Figure S1A). The ability of r84 to specifically block both human and mouse VEGF-induced migration of VEGFR2-expressing endothelial cells (HDMEC, PAE-KDR) but not VEGFR1-expressing endothelial cells (PAE-Flt-1) demonstrates the selectivity of r84 to inhibit VEGF-induced VEGFR2 activity. VEGF binding to VEGFR2 initiates receptor phosphorylation and subsequent phosphorylation of downstream pathway compo- nents such as phospholipase C gamma (PLCc), p38, and the MAP kinase extracellular signal-regulated kinase (ERK1/2). PAE-KDR cells stimulated in vitro with human VEGF (100 ng, two minutes) induced phosphorylation of VEGFR2, PLCy, p38, and ERK (Figure 2B). Human VEGF stimulation of HDMECs induced phosphorylation of PLCc and ERK (Figure 2C). Stimulation of PAE-KDR and HDMEC cells with human VEGF plus 500-fold molar excesses r84 or bevacizumab inhibited phosphorylation of VEGFR2 and downstream targets (Figure 2B, 2C). However, only bevacizumab blocked human VEGF-induced phosphorylation of VEGFR1 in PAE-Flt-1 (Figure 2D). Further, stimulation of PAE- KDR cells with mouse VEGF induced phosphorylation of VEGF2, PLCy, and ERK that was only inhibited by r84 and not by bevacizumab or a control IgG (Figure S1B). This data shows that r84 selectively inhibits human and mouse VEGF PLoS ONE | www.plosone.org 5 August 2010 | Volume 5 | Issue 8 | e12031