Figure 1
Figure 1. Characterization of selected ephrinB2-specific scFvs. (A) Specificity of scFvs for human ephrinB2-Fc (EB2) but not for other related members of the protein family, mouse ephrinB1-Fc (EB1) and human ephrinB3-Fc (EB3) by Western blot. Commercial anti–mouse ephrinB2 polyclonal Ab was used as control. (B) Flow cytometric analysis showing the specificity of B11 and 2B1 to ephrinB2 in native form. HEK293T cells overexpressing human ephrinB1 (left panel), ephrinB2 (middle panel), or ephrinB3 (right panel) were subjected to flow cytometry for detection of B11 and 2B1 binding. As positive controls, specific antisera of each member of the ephrinB family (αeB1, αeB2, and αeB3) were used (lower histograms of each panel). Incubations omitting the specific Abs were used as negative controls. (C) SPR sensorgrams of ephrinB2-specific scFvs, B11 on the left panel and 2B1 on the right, binding to immobilized ephrinB2-Fc are shown. The sensograms were corrected for response differences between the active and reference flow cells. EphrinB2-Fc was immobilized on CM5 chips and scFvs were passed over at concentration ranges noted on each sensorgram, giving affinity constants (KD) of 110nM for B11 and 630nM for 2B1. (D) Inhibition assays using SPR for detection of bound ephrinB2. Serial dilutions of scFv B11 (●) or 2B1 (○) were mixed with 0.2μM ephrinB2-Fc and injected over an immobilized sEphB4 on a CM5 chip. The relative amount of ephrinB2 binding to sEphB4 was measured immediately after injection of each sample and plotted as a function of scFv concentration. Mean values are shown with error bars indicating the SD (n = 3). (E) Activation of EphB4 receptor by tyrosine phosphorylation in HUVECs as a response to interaction with membrane-bound ephrinB2 in the absence or presence of ephrinB2-specific scFv in a cell-based assay. HEK293T cells overexpressing ephrinB2 were overlaid on HUVE cells with or without the corresponding scFv. Total (bottom panel) and phosphorylated EphB4s (top panel) after immunoprecipitation (IP) by Western blot (WB) with the respective Abs are shown. (F) Analysis of EphB4-induced ephrinB2 tyrosine phosphorylation in the absence or presence of B11 and 2B1. HEK293T cells transfected with c-myc–tagged ephrinB2 were treated as indicated and the corresponding cell extracts were immunoprecipitated with anti-c-myc Ab. Total (bottom panel) and phosphorylated (top panel) ephrinB2 were detected by Western blot. The graph on the right showed average levels of tyrosine-phosphorylated ephrinB2, quantified from 3 different immunoblotting experiments. Graph shows normalized results (means ± SD).

Characterization of selected ephrinB2-specific scFvs. (A) Specificity of scFvs for human ephrinB2-Fc (EB2) but not for other related members of the protein family, mouse ephrinB1-Fc (EB1) and human ephrinB3-Fc (EB3) by Western blot. Commercial anti–mouse ephrinB2 polyclonal Ab was used as control. (B) Flow cytometric analysis showing the specificity of B11 and 2B1 to ephrinB2 in native form. HEK293T cells overexpressing human ephrinB1 (left panel), ephrinB2 (middle panel), or ephrinB3 (right panel) were subjected to flow cytometry for detection of B11 and 2B1 binding. As positive controls, specific antisera of each member of the ephrinB family (αeB1, αeB2, and αeB3) were used (lower histograms of each panel). Incubations omitting the specific Abs were used as negative controls. (C) SPR sensorgrams of ephrinB2-specific scFvs, B11 on the left panel and 2B1 on the right, binding to immobilized ephrinB2-Fc are shown. The sensograms were corrected for response differences between the active and reference flow cells. EphrinB2-Fc was immobilized on CM5 chips and scFvs were passed over at concentration ranges noted on each sensorgram, giving affinity constants (KD) of 110nM for B11 and 630nM for 2B1. (D) Inhibition assays using SPR for detection of bound ephrinB2. Serial dilutions of scFv B11 (●) or 2B1 (○) were mixed with 0.2μM ephrinB2-Fc and injected over an immobilized sEphB4 on a CM5 chip. The relative amount of ephrinB2 binding to sEphB4 was measured immediately after injection of each sample and plotted as a function of scFv concentration. Mean values are shown with error bars indicating the SD (n = 3). (E) Activation of EphB4 receptor by tyrosine phosphorylation in HUVECs as a response to interaction with membrane-bound ephrinB2 in the absence or presence of ephrinB2-specific scFv in a cell-based assay. HEK293T cells overexpressing ephrinB2 were overlaid on HUVE cells with or without the corresponding scFv. Total (bottom panel) and phosphorylated EphB4s (top panel) after immunoprecipitation (IP) by Western blot (WB) with the respective Abs are shown. (F) Analysis of EphB4-induced ephrinB2 tyrosine phosphorylation in the absence or presence of B11 and 2B1. HEK293T cells transfected with c-myc–tagged ephrinB2 were treated as indicated and the corresponding cell extracts were immunoprecipitated with anti-c-myc Ab. Total (bottom panel) and phosphorylated (top panel) ephrinB2 were detected by Western blot. The graph on the right showed average levels of tyrosine-phosphorylated ephrinB2, quantified from 3 different immunoblotting experiments. Graph shows normalized results (means ± SD).

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