Fig 2: Receptor binding assays using flow cytometry. (A) HUVECs were incubated with different concentrations of FITC-VGB4 peptide (0.37, 0.55 and 0.74 µM) (green curve) or FITC-scr peptide (0.74 µM) (red curve). Autofluorescence of the cells are reported in violet. (B) and (C) HUVECs were incubated with different concentrations of VGB4 peptide (0.37, 0.55 and 0.74 µM) (blue curve) or scr peptide (0.74 µM) (green curve) for over night in the dark. After adding anti-VEGFR1 and anti-VEGFR2 antibodies, flow cytometric analysis was performed using a BD FACSCalibur Flow Cytometer (see Materials and Methods for details).

Fig 3: VEGF165b inhibition induces VEGFR1-STAT3 signaling and decreases necrosis in eNOS-KO mice in experimental PAD. (A) Necrosis scores in eNOS-KO mice ischemic muscle treated with IgG or VEGF165b-Ab. n = 5, Non-Parametric Mann-Whitney test. (B) TUNEL (shown in green) and CD31 immunostaining (shown in red) to quantify endothelial apoptotic cell death in eNOS-KO mice ischemic muscle treated with IgG or V165b-Ab at day 3 post-HLI. n = 5. Unpaired t-test. (C–E) Immunoblot analysis of V165b, VEGF-A, and pVR1 in VEGFR1 pull-down fractions (VR1-IP); pSTAT3/STAT3 in eNOS-KO mice ischemic muscle treated with IgG or V165b-Ab at day 3 post-HLI. n = 5. Unpaired t-test. * p < 0.05 considered significant. Data Mean ± SEM.

Fig 4: The binding properties of VGB4 to VEGFR1 and VEGFR2 using immunofluorescence. HUVECs were treated for overnight with VGB4, fixed and stained with (A) anti-VEGFR1 –primary antibody which was detected with PE-labeled goat anti-mouse IgG secondary antibody, and (B) anti-VEGFR2 primary antibody which was detected with FITC-labeled rabbit anti-mouse IgG secondary antibody. (C) Graph representing the percent of fluorescence intensity in treated groups compared to control and scr peptide. All data were represented as mean ± SD of three experiments. *P = 0.05, **P = 0.001 versus control. All images were taken by Olympus fluorescence microscope and magnification 400×; bar = 50 µm.

Fig 5: Down-regulation of VEGFR-3 expression and p-Akt levels by MAZ51. The effects of MAZ51 on VEGFR expression and its downstream signal pathway, Akt, were examined in human prostate cancer PC-3 cells by Western blotting. (A) Representative blots of VEGFR-3, p-Akt (Ser473), and total-Akt in the absence and presence of 1 and 3 µM MAZ51 for 48 h in PC-3 cells. (B–D) The expression levels of VEGFR-3 (B), p-Akt (C), and total-Akt (D) in the absence and presence of MAZ51 in PC-3 cells (n = 8). (E,F) Representative blots (E) and the expression levels (F) of VEGFR-1 in the absence and presence of MAZ51 in PC-3 cells (n = 4). (G,H) Representative blots (G) and the expression levels (H) of VEGFR-2 in the absence and presence of MAZ51 in PC-3 cells (n = 4). These expression levels were normalized by that of ß-actin. Data are presented as means ± S.D. *p < 0.05, **p < 0.01 vs. 0 µM MAZ51 (Scheffé’s test following ANOVA).