Fig. 4: EphB4 limits VEGFR2 activation, signaling and turnover.
From: Eph-ephrin signaling couples endothelial cell sorting and arterial specification
a, b Immunoblot detection and quantitation of VEGFR2 phosphorylation (Y951, Y1175) and downstream signaling molecules, as indicated (n = 3 experiments). c pERK1/2 and IB4 staining of P6 retinal vascular front. Lower panels show higher magnification of inverted pERK1/2 channel from depicted insets. Quantitation graph for pERK1/2 immunosignal (n = 3 control and 3 Ephb4iΔEC mice). d Scheme represents effects downstream of EphB4/Fc (B4/Fc) stimulation. e B4/Fc stimulation promotes HUVEC arterialization, while ephrin-B2/Fc (B2/Fc) inhibits it. Average of mRNA expression fold change for investigated genes upon 30 min stimulation of HUVECs with Ctrl/Fc, B4/Fc or B2/Fc (n = 3 experiments). f VEGFR2 and ERK1/2 activation upon B4/Fc stimulation. Immunoblotting and quantitation of relative pVEGFR2 (Y1175) and pERK1/2 levels in stimulated HUVECs treated with VEGFR2 inhibitor (Ki8751) or vehicle (n = 3 experiments). g VEGFR2 signaling is required downstream of ephrin-B2 stimulation to activate DLL4 and HEY1 expression. RT-qPCR for HEY1 and DLL4 (n = 3 experiments). h Increased internalized VEGFR2 in EPHB4 KD cells. Immunolabeling and quantitation of surface (green) and internalized (red) VEGFR2 in siControl and siEPHB4-treated HUVECs (n = 3 experiments). i EphB4 and ephrin-B2 control Epsin1 levels. Immunoblotting and quantitation of Epsin1 protein levels in siControl, siEPHB4 and siEFNB2 HUVECs (n = 3 experiments). P values were calculated by two-way ANOVA (b), two-tailed unpaired t test (c, h) and one-way ANOVA (e–g, i). In vivo experiments were performed with tamoxifen injections at P1-P3 with analysis at P6 (c). Data are presented as mean values ± SEM. Source data are provided as a Source Data file.
