Figure 7: ROBO4 regulates VEGF signalling through UNC5B.
From: The Robo4 cytoplasmic domain is dispensable for vascular permeability and neovascularization
(a) Proposed model for ROBO4 inhibition of VEGFR2 Y951-Src phosphorylation via the UNC5B UPA domain. (b) Western blot analysis of VEGFR2 pY951 and pY1175 in HUVECs transfected with Ctrl or UNC5B siRNA and treated with 3 nM VEGF. (c) Quantifications of blots shown in b. #: VEGF significantly induces pY951 and pY1175 at 5 min. UNC5B siRNA further enhances pY951 but not pY1175. N=5 experiments, Error bars: s.e.m. ***P<0.001, Student's t-test. (d) Schematic diagram showing the engineering of various CD-truncated UNC5B mutants. (e) Western blot analysis and (f) quantifications of pY951 and p-Src in HUVECs with indicated siRNA transfection and adenovirus infection after 3 nM VEGF stimulation. #: VEGF significantly induces pY951 and p-Src at 5 min. Unc5BFL rescues increased pY951 and p-Src in UNC5B silenced cells. N=3 experiments. Error bars: s.e.m. *P<0.05 **P<0.01, Student's t-test. (g) Western blot analysis and (h) quantification of pVEGFR2 in HUVECs with indicated siRNA transfection and adenovirus infection after 3 nM VEGF stimulation. #: VEGF significantly induces pY951 and pY1175 at 5 min. Unc5BΔDD and Unc5BUPA but not Unc5BΔCD and Unc5BΔUPA rescue the excessive VEGFR2 Y951 phosphorylation in UNC5B knockdown cells. None of the constructs affect Y1175 phosphorylation. N=3 experiments. Error bars: s.e.m. *P<0.05; **P<0.01, Student's t-test. (i) Western blot analysis and (j) quantification of pY951 (n=3) in HUVECs with indicated siRNA transfection and adenovirus infection after 3 nM VEGF stimulation. #: VEGF significantly induces pY951 at 5 min. Note that expression of Unc5BFL could not rescue the excessive VEGFR2 Y951 phosphorylation in UNC5B and ROBO4 double siRNA-transfected cells. Error bars: s.e.m. *P<0.05, Welch’s t-test.
