Binding studies by flow cytometry. (A) Presence of CD13 (left panel), α_vβ₃/CD51/CD61 (center), and thrombomodulin (right) on HUVECs as shown by specific AB (curve 1). Curve 2, isotype control. (B) Lack of binding of His-tagged tTF (without NGR) as shown with anti–His-AB (left dot plot). Binding of His-tagged tTF-NGR as shown with anti–His-tag-AB (center). Inhibition of binding of His-tagged tTF-NGR by double amounts of tTF-NGR without His-tag (right). We used single color fluorescence detecting cell-bound His-tagged tTF (left) or His-tagged tTF-NGR (center; step 1) by visualizing with His-tag mouse monoclonal AB (step 2) and then with FITC-labeled goat anti–mouse Ig (step 3). In the right dot plot, cells were first incubated with tTF-NGR without His-tag and were then incubated with His-tagged tTF-NGR (see step 1 above) with the identical visualization sequence as above (see steps 2 and 3). (C) Binding of AB to CD13 (i left), α_vβ₃ (ii left) binding sites and thrombomodulin control site (iii left). Replacement of AB-binding by 150 μg/mL tTF-NGR to CD13 (i, right), α_vβ₃ (ii right) in contrast to tTF (i,ii center). No competition of 150 μg/mL tTF-NGR with AB-binding to thrombomodulin (iii right). Dot plots with single color show fluorescence intensity on x-axis. The y-axis titled “FL2-Height” was not occupied in all dot plots using the “FITC-protocol” (see “Methods”) and the y-axis (FL-1) using the “PE-protocol” (see “Methods”) was occupied with mouse IgG 1 FITC to yield a better visualization of the cell populations.