To characterize VLR affinity for bEnd.3 ECM, dilutions of purified VLR were incubated with ECM. P1C10 VLR was diluted using a sixfold dilution scheme from 10 μM to 0.21 nM and then incubated with bEnd.3 ECM. Wells were washed five times with PBS + 0.05% Tween 20 and then inubcated with anti-myc 9E10, 1:750, (BioLegend) and goat anti-mouse IgG modified with HRP. Wells were washed seven times with PBS + 0.05% Tween 20 and then incubated with TMB substrate. Reaction was stopped after 15 min by acidification with 1 M HCl. Absorbance at 450 nm was quantified, and data were fit to a one-site equilibrium binding model to determine Kd as previously described (43). The amino acid sequence for VLR P1C10 is ACPSQCSCDQTTVKCHSRRLTSVPAGIPTTTKILRLYSNQITKLEPGVFDHLVNLEKLYISWNQLSALPVGVFDKLTKLTHLSLGYNQLKSVPRGAFDNLKSLTHIWLLNNPWDCECSDILYLKNWIVQHASIVNLQGHGGVDNVKCSGTNTPVRAVTEASTSPSKCP.

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