Fig. 1: Generation of a Tie2-activating mouse monoclonal antibody.
a Schematic of the domain structure of the Tie2 receptor. Ig immunoglobulin-like domain, EGF epidermal growth factor-like domain, Fn fibronectin type III domain. b Immunoblot detection of Tie2 downstream signaling (Akt and p-Akt) in HUVECs stimulated with each of seven purified Tie2-activating antibodies (11C4, 4A4, 4C2, 5F4, 3B2, 3E12, and 3H7; 0.1, 1, and 10 μg/ml) for 30 min. The selected 3H7 clone is colored in red. No treatment for negative control (Ctrl) and COMP-Angpt1 (CA1, 1 μg/ml) for positive control. c Concentration-dependent Tie2 phosphorylation upon treatment of HUVECs with 3H7 (0.02, 0.1, 0.5, 2.5, 10, 50 μg/ml). Immunoblot (top) and densitometric analyses (bottom) of p-Tie2/Tie2 ratios are shown. Data from three independent experiments (n = 3) were analyzed and expressed as mean ± SD (*P = 0.0104, ***P < 0.001 vs. control). P values by one-way ANOVA test followed by Dunnett’s multiple comparisons test. ns, not significant. d Representative confocal images of HUVECs showing hTAAB-induced Tie2 translocation to cell–cell contacts (top) and hTAAB-induced nuclear clearance of FOXO1 (bottom). Serum-starved HUVECs were treated with hTAAB (10 μg/ml) or COMP-Angpt1 (1 μg/ml) for 30 min. Goat anti-human Tie2 and Alexa Fluor 488-conjugated donkey anti-goat antibodies were used for Tie2 visualization, and rabbit anti-FOXO1 and Alexa Fluor 594-conjugated donkey anti-rabbit antibodies were used for FOXO1 visualization. Scale bars, 20 μm. DAPI, 4’, 6-diamidino-2-phenylindole. Similar results were observed in three independent experiments. e Binding kinetics of hTAAB for human (left) or mouse (right) Tie2 determined by SPR and BLI analysis, respectively. The equilibrium dissociation constant (KD, M) was calculated as the ratio of off-rate to on-rate (koff/kon). Kinetic parameters were determined with the global fitting function of Biacore Insight Evaluation Software using a 1:1-binding model.
