Figure 2.

(A) Flow-cytometric analysis of binding of Z_VEGFR2-Bp₂ to different cell lines, MS1, PC-3 and GL261. Binding of Z_VEGFR2-Bp₂-ABD to cells was detected by Alexa Fluor (AF) 647-labeled HSA. AF-647 labeled HSA was included as negative control. Ramucirumab (anti-hVEGFR2) and DC101 (anti-mVEGFR2) were included as controls for binding to human (PC-3) or mouse (MS1 and GL261) cell lines. Blue histograms indicate affibody/antibody samples, and red histograms are negative controls. The experiment was performed in duplicate. (B) In vitro binding specificity of [¹¹¹In]In-NODAGA-Z_VEGFR2-Bp₂ tested on MS1 cells in the presence or absence of non-labeled Z_VEGFR2-Bp₂, VEGFA, or anti-mVEGFR2 antibody DC101. The cell-associated activity is presented as a percentage of the total added activity (average value from three cell dishes ± SD). (C) Binding and internalization of [¹¹¹In]In-NODAGA-Z_VEGFR2-Bp₂ by MS1 cells. Data are presented as average values from three cell dishes ± SD. Error bars might not be visible because they are smaller than point symbols. (D) Real-time binding data of the [¹¹¹In]In-NODAGA-Z_VEGFR2-Bp₂ interaction. Arrows indicate the concentration change. (E) Binding of [¹¹¹In]In-NODAGA-Z_VEGFR2-Bp₂ to MS1 cells after 2 h treatment with 25 nM 17-DMAG. An equal amount of cells per culture dish was seeded, and equal [¹¹¹In]In-NODAGA-Z_VEGFR2-Bp₂ activity was added to each cell culture dish. The data are presented as the average value for three cell dishes ± SD. Uptake by treated cells was significantly (p < 5×10^-6) lower than by untreated controls. (F) Survival of MS1 cells after treatment with 25 nM 17-DMAG over 48 h. The data are presented as the average value for three cell culture flasks ± SD.