Fig. 6.

ROBO1 guides ENDOA2-mediated endocytosis via srGAP1. a ENDOA2 immunoprecipitation in HUVECs cultured in full medium and western-blot analysis of the indicated proteins. VEGFR2, ROBO1, srGAP1, and ENDOA2 expression from the total cell lysate are shown as loading controls (input). IP ENDOA2 (−): cell lysate incubated with beads alone; IP ENDOA2 (+): cell lysate incubated with anti-ENDOA2 antibody + beads. b Confocal images of HUVECs stained for VEGFR2, ENDOA2, and srGAP1 after 2′30″ VEGF (1.5 nM) or SLIT2 (3 nM) stimulation. Boxed areas are magnified to highlight VEGFR2/ENDOA2/srGAP1 pixel overlap. c SIM images of the lamellipodia of Ctrl and srGAP1 siRNA treated HUVECs stained for ENDOA2 and VEGFR2 after VEGF or SLIT2 stimulation (1.5 or 3 nM for 2′30″). Overlapping pixels between VEGFR2/ENDOA2 fluorescent signals are shown in white. Boxed areas are magnified to highlight VEGFR2/ENDOA2 pixel overlap. d Quantification of pixel overlap between VEGFR2 and ENDOA2 (N = 10 cells per group, three independent experiments; Mann–Whitney U test: *P < 0.05, **P < 0.01). e Antibody feeding assay to assess VEGFR2 internalization in response to VEGF or SLIT2 (3 or 6 nM) in Ctrl and srGAP1 siRNA silenced HUVECs. f quantification of internalized VEGFR2 fluorescent intensity (N = 3–6 independent experiment, at least 10³ cells analyzed per experiment; one-way ANOVA: **P < 0.01). g GFP immunoprecipitation of ROBO1/2 silenced cells transduced with the indicated constructs and western blot analysis of the indicated proteins. IP GFP (−): cell lysate from ROBO1/2 silenced cells transduced with ROBO1WT-GFP construct incubated with beads alone. h Golgi orientation of ROBO1/2 silenced HUVECs transduced with the indicated constructs (N = 3 experiments, one-way ANOVA: ns P > 0.05, *P < 0.05, **P < 0.01). Error bars represent mean ± s.e.m. Scale bars: b 2 μm, c 2 μm, e 20 μm