Fig. 5

ROBO1 promotes VEGF-induced ENDOA2-mediated VEGFR2 endocytosis. a VEGFR2 immunoprecipitation in HUVEC after VEGF (1.5 nM for 2′30″) or SLIT2 (3 nM for 2′30″) stimulation, and western blot for ROBO1 and VEGFR2. ROBO1 and VEGFR2 expression from the total cell lysate are shown as loading controls (input). b SIM image of HUVECs stained for VEGFR2, ENDOA2, and ROBO1 after VEGF stimulation (1.5 nM for 2′30″). Right panel is a higher magnification of the boxed area in the left panel. c SIM images of the lamellipodia of Ctrl and ROBO1/2 siRNA treated HUVEC stained for ENDOA2 and VEGFR2 after VEGF stimulation (1.5 nM for 2′30″). Overlapping pixels between VEGFR2/ENDOA2 fluorescent signals are shown in white. Boxed areas are magnified to highlight VEGFR2/ENDOA2 proximity. d Quantification of pixel overlap between VEGFR2 and ENDOA2 fluorescent signals (N = 8 cells per group analyzed from three independent experiments; Mann–Whitney U test: ***P < 0.01). e Western-blot analysis of VEGF-induced VEGFR2 internalization (3 nM) from cell surface biotinylation assay in Ctrl or ROBO1/2 siRNA silenced HUVECs. VEGFR2, ROBO1, and ACTIN expression from the total cell lysate are shown as loading controls (input). Surf: surface expression. f Quantification of internalized VEGFR2 normalized to VEGFR2 surface expression before stimulation. (N = 7 independent experiments; two-way ANOVA: ns P > 0.05, **P < 0.01). g Antibody feeding assay to assess VEGFR2 internalization in response to VEGF (3 nM) in Ctrl and ROBO1/2 siRNA silenced HUVECs. Quantification of internalized VEGFR2 fluorescent intensity (right panel) (N = 4 independent experiments, at least 10³ cells analyzed per experiment; Mann–Whitney U test: *P < 0.05). h Golgi orientation in tip cells from Robo1,2^+/+ and Robo1,2^−/− P5 retinas. (N = 3 retinas per group, at least 50 tip cells per retina were quantified, two-way ANOVA: *P < 0.05, ***P < 0.001, ns P > 0.05). Error bars represent mean ± s.e.m. Scale bars: b left panel 5 μm, b right panel 1 μm, c 2 μm, g 20 μm