The Rab5 GEF RIN2 protects VEGFR2 from degradation and promotes angiogenic sprouting. a Pie diagram showing the relative mRNA levels of Rab5 GEFs in HUVECs. b Quantification of cell-surface levels of VEGFR2, as assessed by flow cytometry, in HUVECs transduced with shRNAs against the indicated GEFs. Values represent mean fluorescence intensities + SEM of 3–5 independent experiments, expressed relative to sh_Ctrl cells. c Western blot analysis of VEGFR2 and RIN2, using α-tubulin as a loading control. d Quantification of VEGFR2 levels from Western blots. Values represent the means + SEM of 3 individual experiments relative to sh_Ctrl. e Quantification of VEGFR2 degradation from Western blots in sh_Ctrl and sh_RIN2 cells that were starved overnight and subsequently either maintained in growth factor-free medium or stimulated with 50 ng/ml VEGF. Bars represent means + SEM of 3 independent experiments, expressed relative to sh_Ctrl cells at t = 0. f Representative images (maximum projections from confocal z-stacks) showing sprouting of sh_Ctrl and sh_RIN2 HUVECs. Staining shows F-actin (magenta) and nuclei (cyan). Scale bar, 75 μm. g Quantification of the average number of large sprouts/bead for sh_Ctrl and sh_RIN2 cells. Values represent means + SEM of 3 independent experiments (20 beads per experiment). Quantification of h total network length and i average total number of sprouts in sh_Ctrl (n = 18 beads) and sh_RIN2 cells (n = 16 beads). A representative experiment is shown
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