Figure 1.

EVA-R expressed in mammalian cells binds CC chemokines and inhibits their function.A and B, analytical reversed-phase high performance liquid chromatogram (A) and analytical size-exclusion chromatogram (B) of purified EVA-R. C, Coomassie blue–stained SDS-polyacrylamide gel showing purified EVA-R before and after PNGase F treatment: lane 1, protein molecular weight markers (labeled in kDa); lane 2, EVA-R after purification by size-exclusion chromatography; lane 3, purified EVA-R after treatment with PNGase F; lane 4, PNGase F alone. D, representative surface plasmon resonance sensorgrams showing binding of immobilized EVA-R to six CC chemokines (CCL3, CCL4, CCL13, CCL14, CCL15, and CCL18) but not to other CC or any CXC, CX₃C, or XC chemokines, at 500 nM concentration. E, representative surface plasmon resonance sensorgrams showing binding of immobilized EVA-R to five different concentrations of the indicated chemokines (31.25, 63.5, 125, 250, and 500 nM). F, binding affinities of EVA-R for CC chemokines, measured by SPR (mean ± SD from three independent experiments). G, concentration response curves showing the inhibition of chemokines CCL3 and CCL4 by EVA-R. FlpInCHO cells stably expressing CCR5 and transfected with the cAMP biosensor CAMYEL, were treated with coelenterazine-H (5 μM, 10 min), followed by forskolin (10 μM, 10 min), followed by CCL3 (60 nM) or CCL4 (80 nM), either alone or pre-incubated with the indicated concentrations of EVA-R. Inhibition of forskolin-induced cAMP production was detected 10 min after chemokine addition. Data are presented as mean ± SEM from three independent experiments. cAMP, cyclic AMP; SPR, surface plasmon resonance.