Immunoblotting analysis

Whole cell protein lysates were prepared from HUVEC using CelLytic reagent (Sigma). Immunoblotting of cell lysates was performed according to standard conditions. Immunoblots were labelled with the following primary antibodies: anti-Akt (11E7) (4685, 1:1,000, Cell Signaling Technology), anti-phospho (S473)-Akt (9271, 1:1,000, Cell Signaling Technology), anti-ERG (sc353, 1:500, Santa Cruz Biotechnology), anti-ERG (ab133264, 1:1,000, Abcam), anti-Dll4 (1:500, R&D systems), anti-GAPDH (MAB374, 1:10,000, Millipore), anti-Jag1 (sc-6011, 1:1,000, Santa Cruz Biotechnology), anti-NICD/cleaved Notch1 (Val1744) (2421, 1:500, Cell Signaling), anti-Tie2 (D9D10) (7473, 1:1,000, Cell Signaling). Primary antibodies were detected using fluorescently labelled secondary antibodies: goat anti-rabbit IgG DyLight 680 and goat anti-mouse IgG Dylight 800 (Thermo Scientific). Detection and quantification of fluorescence intensity were performed using an Odyssey CLx imaging system (LI-COR Biosciences, Lincoln) and Odyssey 2.1 software. In some instances, HRP-conjugated secondary antibodies were used for chemiluminescence detection and protein levels were quantified by densitometry and normalized against loading controls. See Supplementary Fig. 10 for the uncropped immunoblots.