Impact of ligand binding on VEGFR1, VEGFR2, and NRP1 localization in human endothelial cells
Fig 2
Experimental measures of whole-cell and surface levels of VEGFR1, VEGFR2, and NRP1 in HUVECs.
A, B, Western blot of total HUVEC lysates treated as indicated for 0.25–4 hours with 50 µg.mL-1 of VEGF165a (A) or PLGF1 (B) and stained for VEGFR1, VEGFR2 and NRP1. Representative of n = 3 replicates. C, D, Quantification of whole cell receptor levels shown in A,B. E, F, Western blot of Biotin labeling assay to measure surface and internal VEGFR1, VEGFR2, and NRP1 levels in HUVECs; for 1 hour and 4 hours with 50 µg.mL-1 of VEGF165a or PLGF1 and stained for VEGFR1, VEGFR2 and NRP1. Representative of n = 3 replicates. Western blot showing the effect on VEGFR1, VEGFR2 and NRP1 levels of depletion of both Rab4a and Rab11a for 1 hour and 4 hours ligand treatment; Representative of n = 3 replicates. Control conditions in E and F include nontargeting siRNA (siNT). Reagents used for the experiments are detailed in S19 Table. G, H, Quantification of whole cell receptor levels shown in E, F. The trend of reduced whole cell VEGFR2 following VEGF165a administration observed in A and C is consistent with significant differences (by single factor ANOVA analysis, *, p < 0.01; **, p < 0.0001) for surface and whole cell VEGFR2 levels at 1 hour and 4 hours post-VEGF165a addition compared to no ligand addition, observed in E-H.