Fig 2: Analysis of the VEGFR2/VE-cadherin/Src complex in A2780 and CAOV3 tumors treated with or without siRNA targeting ANGPTL4. a Immunoprecipitation (IP) of vascular endothelial growth factor receptor 2 (VEGFR2) and immunoblotting (IB) of VE-cadherin, Src kinase phosphorylated at tyrosine 416 (Src pY416), Src, and VEGFR2. Total lysates were immunoblotted for VEGFR2 phosphorylated at tyrosine 949 (VEGFR2 pY949), VEGFR2, VE-cadherin, Src pY416, Src, and GAPDH; n = 3 independent experiments per group. b VE-cadherin/VEGFR2 ratio in immunoprecipitates from A2780 and CAOV3 tumors. c The Src pY416 level normalized to that of GAPDH or Src and the Src/GAPDH ratio were determined in immunoblots of A2780 and CAOV3 total lysates. d The VEGFR2 pY949 level normalized to that of GAPDH or VEGFR2 and the VEGFR2/GAPDH ratio were analyzed in total lysates by immunoblotting. e VE-cadherin/GAPDH ratio in immunoblots of A2780 and CAOV3 total lysates. **p < 0.01, ***p < 0.001 for the indicated comparisons. ns indicates no significance. The data are presented as the mean ± SEM values

Fig 3: Embryonic and postnatal vascular development in Vegfr2 Y1212F/Y1212F C57Bl/6 mice APartial embryonic lethality after E11.5. Chi-square, ****P < 0.0001. E11.5 n = 114 embryos, E14.5 n = 70 embryos, adult n = 314 born pups.BHindbrain vessel morphology. Isolectin B4 (IsoB4; white), ERG (red), and EdU incorporation (green). EdU signal was masked with ETS-related gene (ERG) signal. Scale bar, 50 µm.CHindbrain vessel density. Isolectin B4 vessel area normalized to wild-type (WT) mean; one dot is two fields/mouse; error bars: SD; unpaired t-test, *P < 0.05. n = 18–20.DHindbrain EC proliferation. EdU incorporation in ERG-positive ECs (double-positive cells, yellow in B). One dot is two fields/mouse; error bars: SD; unpaired t-test, *P < 0.05. n = 7–12.EReduced weight gain in the adult. Error bars: SD; 2-way ANOVA P = 0.0009; Sidak's multiple comparison test, **P < 0.01. n = 13–20 mice.FP6 retina vessel morphology. Isolectin B4 (IsoB4) in black; the outer 20% of the vascularized area in pink. Scale bar, 250 µm.G–JVessel parameters in the P6 developing retina. (G) Radial outgrowth (distance between optic nerve and vascular front). (H) Vascularized area. (I) Vessel area in the outer 20% of the retinal vasculature; pink in (F) (vessel area normalized to vascularized area). Each dot represents one retina/mouse; n = 3–4. (J) EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents one retina/mouse; error bars: SD. n = 8–9.

Fig 4: Specific binding of VEGFR2 Y1212 to GRB2 and PI3Kp85 A, BImmunoblot showing VEGFR2 phosphorylation on pY1212, pY949, and pY1173. (A) Anti-VEGFR2 immunoprecipitates (IP) from lung lysates from wild-type (WT) and Vegfr2 Y1212F/Y1212F FVB mice, tail-vein-injected with PBS or VEGF for 1 min. (B) Quantification as percentage of phosphoprotein level normalized to total VEGFR2. Each lane (A) and each dot (B) represent one individual mouse. Sidak's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001. n = 3.CSummary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate. Ten proteins (red oval) were selected for further analysis. PSM: peptide-spectrum match. Area: average area of spectra for the three most abundant peptides in arbitrary units.DBlot for peptide-enriched GRB2 and PI3Kp85. HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85.E–GVEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes in isolated (i) ECs. (E) PLA for VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes (black puncta) in iECs from FVB WT and Vegfr2 Y1212F/Y1212F lungs. Hoechst 33342 show nuclei (gray). Scale bar, 20 µm. (F, G) Fold increase over PBS-treated sample; each dot represents the mean of six fields. Two-way ANOVA P = 0.0206 (F), P = 0.0368 (G); Sidak's multiple comparison test, *P < 0.05. n = 3 experiments.HphosphoERK1/2 and phosphoAkt immunostaining (green) of iECs from FVB WT and Vegfr2 Y1212F/Y1212F lungs. Hoechst 33342 (blue) shows nuclei. Arrows indicate nuclear accumulation of phosphoERK1/2 and phosphoAkt, respectively. Scale bar, 20 µm.INuclear accumulation expressed as phosphoERK1/2-positive nuclear area normalized to the total phosphoERK1/2 area by field. Each dot represents 1 field. Min, minutes of stimulation. Two-way ANOVA P = 0.0248; Sidak's multiple comparison test, *P < 0.05, **P < 0.01. n = 3 experiments.JNuclear accumulation expressed as fold increase of nuclear phosphoAkt area in Vegfr2 Y1212F/Y1212F over PBS-treated WT iECs, normalized to the number of nuclei. Each dot represents 1 field. Min, minutes of stimulation. Unpaired t-test, *P < 0.05. n = 3 experiments.KImmunoblot for phosphoERK1/2, total ERK1/2, phosphoAkt, and total Akt on lung lysates from WT and Vegfr2 Y1212F/Y1212F FVB mice tail-vein-injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse.LQuantification of phosphoERK1/2/total ERK1/2 expressed as fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA P = 0.0025; unpaired t-test, *P < 0.05. n = 3 experiments.MQuantification of phosphoAkt/total Akt expressed as fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA P = 0.0003; unpaired t-test, *P < 0.05. n = 3 experiments. Source data are available online for this figure.

Fig 5: Induction of pERK1/2, pAkt, and human MYC in WT and VEGFR2 pY1212 conditions AphosphoERK1/2 immunostaining (black) of iECs from FVB wild-type (WT) and Vegfr2 Y1212F/Y1212F lungs. Arrows indicate nuclear accumulation of phosphoERK1/2. Scale bar, 20 µm.BphosphoAkt immunostaining (black) of iECs from FVB WT and Vegfr2 Y1212F/Y1212F lungs. Note: Arrows indicate nuclear accumulation of phosphoAkt in response to VEGFA. Scale bar, 20 µm.CTamoxifen-induced human Myc gene expression in the E11.5 MycOE/Cre+ embryos. Quantitative PCR analysis of human MYC mRNA in E11.5 Myc overexpressing (MycOE/Cre+) and MycOE/Cre- embryos after 100 µl of 4OHT-tamoxifen injection at E9.5. Unpaired t-test, *P < 0.05. n = 8–9 embryos.