Normal rabbit IgG fraction was used as control primary antibody

PDZ binding motif or the cleavage sites should cautiously be interpreted. Materials and Methods Plasmids and Viruses Full length human JAG1 cDNA with a c-terminal myc tag in the pCMV-ENTR vector was from OriGene and shuttled by 9537826 EcoRI-EcoRV into pENTR3c. The intracellular domains of murine Dll1, Dll4 and Jag1 were PCR amplified and ligated into pCS2p and pCS2-HA-Citrine, which leads to expression of fusion proteins with the HA tag and the fluorescent Citrine at the aminoterminal site. The INK1117 ligand-ICD without tag and the Citrine-ligand-ICD cassettes were released with HindIII -EcoRI and transferred into pENTR3c. The Bi-Directional Notch Signaling in the Endothelium adenoviral NOTCH1-ICD construct was described before. Adenoviruses were generated in HEK293 cells using the ViraPower adenoviral Expression Systems kit. Semiconfluent endothelial cells were transduced with multiplicity of infection of 50. Cell Culture Primary human endothelial cells from umbilical veins were purchased from PromoCell and cultured in endothelial growth medium with supplements and 10% FCS. Only cells of passages 26 were used for experiments. harvested from the other three wells. Remaining DNA was digested with DNase I. RNA quality was determined on an Agilent 2100 Bioanalyzer. Screening on Illumina Sentrix human WG-6 v3.0 bead chips was performed as described. Raw data were quantile normalized and analyzed by Bead Studio 3.1.3 software with the Genome Studio Plugin. Transcripts with detection p-values smaller 0.05 were selected. Expression changes were calculated with the 22891655 Illumina Custom error model including a Benjamini and Hochberg algorithm for multi-testing corrections. Probes with p-values smaller 0.001 were filtered. The complete data set of the NOTCH1-ICD vs. GFP experiment can be found in the NCBI GEO database. Endothelial Cell Proliferation, Migration, Adhesion and Sprouting Cell proliferation was measured by the incorporation of 5bromodeoxyuridine. HUVEC were seeded into in 96-well plates 24 h after viral transduction. BrdU was added for 12 h and DNA incorporation was quantified by ELISA. Chemotactic cell migration was determined in a modified Boyden chamber. The filter was coated with collagen-I. HUVEC were seeded on top of the membrane and 25 ng/ml VEGF or FGF-2 was added to the lower chamber as a chemoattractant. After 4 h incubation the cells at the lower side of the filter were fixed with EtOH, stained by Giemsa solution and counted. Cell adhesion was detected 48 h after viral infection. Cells were seeded in 96-well plates. The plates were either untreated or coated with collagen-I, 0.2% gelatin or fibronectin. The plates were incubated for 30 min and then shaken for 10s in a standardized procedure. After washing with medium, the adherent cells were fixed with 4% paraformaldehyde, washed and stained with crystal violet for 10 min. Cells were washed again with water, dried and lysed with 2% SDS. Adhesion was quantified by measuring the absorption of the solution at 550 nm. The endothelial spheroid sprouting angiogenesis assay was performed as described. In short, HUVEC were suspended in 20% methocel and cultured as hanging drops overnight. Thereby, cell spheroids formed. The spheroids were washed and embedded in collagen. Medium lacking growth factors or containing VEGF or FGF-2 was added to the collagen beds. Capillary-like structures were assessed 24 h later. Ten spheroids per condition were analyzed and the assay was repeated three times with diff