Anti dll4

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.

The antibodies used for FACS analysis were: mouse IgG2b-FITC, goat IgG-PE, anti-Jagged1-FITC, anti-Dll3-PE (all from R&D System, Minneapolis, MN), mouse IgG2a-PE, mouse IgG1κ-PE, mouse IgG1-Alexa Fluor 488,anti-Notch1-PE, anti-Notch2-PE, anti-Notch3-PE, anti-Notch4-PE, anti-Dll1-PE, anti-Dll4, anti-Bax-Alexa Fluor 488 (all from Biolegend, San Diego, CA) and rabbit anti-Bcl-2-FITC (DAKO). For leukemia cell identification we used anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP and anti-CD117-APC (all from MiltenyiBiotec, Germany). The antibodies employed for western blot analysis anti-Notch2, anti-Notch4 were from Santa Cruz (Biotechnology, Dallas, TX), anti-GAPDH and HRP conjugated secondary antibodies against mouse, rabbit or goat were from Sigma Aldrich. All the other antibodies used for Western blot were from Cell Signalling. Neutralizing antibodies,all used at a final concentration of 5 μg/ml, were: anti-Notch1, anti-Notch3,anti-Jagged1, anti-Jagged2, anti-Dll1 and anti-Dll4 (R&D Systems); anti-Notch-4 (Santa Cruz Biotechnology); anti-Dll3 (CST, Boston, MA). Recombinant human Jagged-1 and Jagged-2 were from R&D System. GSI-IX (DAPT) was purchased from Stemgent (Cambridge, MA) GSI-XII and SAHM1 were from Merck Millipore (Darmstadt, Germany). Cytarabine (Ara-C), Etoposide (Eto) and Idarubicin (Ida) were provided by Pharmacy Unit of the University Hospital of Verona.