Intracellular Staining by Flow Cytometry
|Detection of Notch‑1 in RPMI 8226 Human Cell Line by Flow Cytometry. RPMI 8226 human multiple myeloma cell line was stained with Mouse Anti-Mouse Notch‑1 PE‑conjugated Monoclonal Antibody (Catalog # IC5267P, filled histogram) or isotype control antibody (Catalog # IC002P, open histogram). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
Notch-1 is a 300 kDa type I transmembrane glycoprotein that is one of four Notch homologues involved in developmental processes (1-3). Notch signaling is important for maintaining stem cells and inducing differentiation, especially in the nervous system and lymphoid tissues (2-4). Notch can specify binary cell fates. For example, it promotes T cell over B cell development from a common precursor (2). Mouse Notch-1 is synthesized as a 2531 amino acid (aa) precursor that contains an 18 aa signal sequence, a 1707 aa extracellular domain (ECD) with 36 EGF-like repeats and three Lin-12/notch repeats (LNR), a 21 aa transmembrane (TM) segment and a 785 aa cytoplasmic domain that contains six ankyrin repeats, a glutamine-rich domain and a PEST sequence. The 11th and 12th EGF-like repeats, that bind ligands such as Jagged and Delta-like families in humans, correspond to aa 412-488 in mouse Notch-1 (6). Elongation of O-linked fucose chains by Fringe family members at a site within this region can inhibit the interaction of Notch with Jagged ligands, thereby promoting Delta-like ligand interactions (7). The Notch-1 receptor undergoes post-translational furin-type proteolytic cleavage, generating a heterodimer through the interaction of a hydrophobic area C-terminal to the LNR on the extracellular region with the transmembrane/cytoplasmic portion (8, 9). Upon ligand binding, additional sequential proteolysis by TNF-converting enzyme (ADAM17) and the presenilin-dependent gamma -secretase results in the release of the Notch intracellular domain (NICD) which translocates into the nucleus, activating transcription of Notch-responsive genes (10).