Due to a direct effect of the treatment, as well as by the activation of several mediators of the immune response. The evolutionarily conserved Notch signaling pathway mediates direct cell-to-cell communication and regulates numerous developmental processes. Notch genes encode transmembrane proteins that act at the surface of a cell as receptors for transmembrane proteins encoded by the Delta and Serrate in mammals) genes. NOTCH as well as its ligands have a gene-specific number of epidermal growth factor-like repeats in their extracellular domains that are critical for receptor-ligand interaction. Upon ligand binding, the intracellular portion of NOTCH is proteolytically released, translocates to the nucleus, and by BAY 43-9006 284461-73-0 binding to a transcriptional regulator of the CSL family, activates transcription of target genes. Posttranslational modification of NOTCH by O-fucose is essential for Notch signaling both in Drosophila and mammals. Protein O-fucosyltransferase 1, which is encoded by Ofut1 in Drosophila and Pofut1 in mammals, adds O-fucose to Ser or Thr residues that are part of a consensus motif in certain EGF repeats of NOTCH. O-Fucose residues on EGF repeats can be further modified by Fringe proteins, fucose-specific b1,3 N-acetylglucosaminyltransferases that act in the trans-Golgi. Notch modification by Fringe affects the ability of ligands to activate Notch receptors in a context-dependent manner, but O-fucosylation was dispensable for Notch activity during embryonic neurogenesis in Drosophila. In addition to providing the substrates for Fringe proteins, POFUT1 appears to influence Notch function in several ways. Analysis of OFUT1 mutants in Drosophila led to the conclusion that OFUT1 has a chaperone activity distinct from its fucosyltransferase activity that assists in Notch folding and cell-surface presentation. Another study suggested that Drosophila OFUT1 also acts extracellularly and regulates Notch endocytosis thereby maintaining stable Notch presentation at the cell surface. In mammalian cells in culture and in haematopoietic cells in mice loss of POFUT1 did not prevent surface expression of Notch receptors but caused reduced ligand binding and Notch activity, whereas in the paraxial mesoderm of mice lacking POFUT1 Notch1 was reported to accumulate in the ER. These apparent differences notwithstanding, POFUT1 is clearly required for normal Notch function. EGF repeats of the ligands also contain recognition sites for POFUT1 that are O-fucosylated. OFUT1 appears to be dispensable for folding or function of ligands in Drosophila, but the significance of O-fucose modification or fucosyltransferaseindependent functions of POFUT1 for the activity and localization of vertebrate ligands is unclear. Here, we focus on the murine Notch ligand DLL1. We show that EGF repeats 3, 4, 7, and 8 are stoichiometrically modified with O-fucose at the predicted consensus sites. DLL1 variants in which the Ser or Thr residues in the consensus sites were replaced with Ala and Val residues, respectively accumulated intracellularly in addition to their cell surface localization.