Fbxw7 Controls Angiogenesis by Regulating Endothelial Notch Activity
Figure 4
Fbxw7 controls endothelial Notch signaling.
Protein extracts prepared from P6 control (lanes 1 & 3 from the left) and Fbxw7iECKO lungs (lanes 2 & 4) showed increased amounts of active Notch1 (NICD-Val1744) and reduced VEGFR3 protein (A). VE-Cadherin (Cdh5) was used for normalization. Relative Fbxw7 mRNA expression in HUVECs transfected with siFbxw7 relative to siControl siRNAs (n = 3 per group) (B). Human GAPDH was used for normalization. VEGF-A stimulation (50 ng/ml for the indicated time periods) led to a transient accumulation of active Notch (NICD) in HUVECs transfected with siControl. NICD levels were upregulated after Fbxw7 silencing (C). Western blot data (band intensity in pixels) was quantified with ImageJ; the graph shows average values from 3 independent experiments (D). Alpha-tubulin was used for normalization. Confocal images showing the pronounced reduction of VEGFR3 protein (green) in the endothelium (Isolectin B4; red) of the P6 Fbxw7iECKO retina (E). Scale bar is 100 µm. Quantitative analysis (with Volocity 5; n = 3 for each group) of image data confirmed reduced VEGFR3 levels (number of pixels) in mutant ECs (F). Error bars (in D and F) indicate SEM. P values are indicated as ** (<0.001) and * (p<0.05).