Figure 5: Robo4ΔCD affects VEGF signalling through VEGFR2 Y951.
From: The Robo4 cytoplasmic domain is dispensable for vascular permeability and neovascularization
(a) Western blot analysis of VEGFR2 and Src phosphorylation in HUVECs transfected with Ctrl or ROBO4 siRNAs and treated with 3 nM VEGF. (b–d) Quantifications of blots shown in a. #: VEGF significantly induces VEGFR2 pY951, pY1175 and p-Src at 5 min. ROBO4 siRNA further increases pY951 and p-Src, but not pY1175. N=4–6 experiments. Error bars: s.e.m. NS, not significant; *P<0.05; **P<0.01, Student's t-test. (e) Western blot analysis of pVegfr2 and p-Src in MLECs from Robo4+/+ and Robo4−/− mice after 6 nM VEGF stimulation. (f–h) Quantifications of blots shown in e. #: VEGF significantly induces Vegfr2 pY949 and pY1173. Robo4−/− cells show further enhanced pY949 and p-Src, but not pY1173. N=3 experiments. Error bars: s.e.m. NS, not significant; *P<0.05, Student's t-test. (i) Western blot and (j) quantification of pY951 in HUVECs with indicated siRNA transfection and adenovirus infection after 3 nM VEGF stimulation. #: VEGF significantly induces VEGFR2 pY951 at 5 min. ROBO4FL rescues increased pY951 in ROBO4 silenced cells. N=4 experiments. Error bars: s.e.m. **P<0.01, Student's t-test. (k) Western blot analysis of pY951 (n=6) and p-Src (n=4) and the corresponding quantifications (l) in HUVECs with indicated siRNA transfection and adenovirus infection after 3 nM VEGF stimulation. #: VEGF significantly induces VEGFR2 pY951 and p-Src at 5 min. Robo4ΔCD restores pY951 and p-Src in ROBO4 silenced cells to Ctrl levels. Error bars: s.e.m. *P<0.05, **P<0.01, Student's t-test. (m) Western blot and (n) quantification of pVegfr2 in MLECs from Robo4−/−;Stg and Robo4−/−;Robo4ΔCD mice after 6 nM VEGF stimulation. Note decreased Y949 but unchanged Y1173 in Robo4−/−;Robo4ΔCD. N=5 experiments. Error bars: s.e.m. NS, not significant; *P<0.05, Student's t-test. (o) Schematic of ROBO4 and Robo4ΔCD effects on VEGF signalling through VEGFR2.
