|Detection of Mouse and Rat Notch‑2 by Western Blot. Western blot shows lysates of Balb/3T3 mouse embryonic fibroblast cell line and rat embryonic hippocampal neuron tissue. PVDF Membrane was probed with 2 µg/mL of Sheep Anti-Mouse/Rat Notch‑2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5196) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for Notch‑2 at approximately 275 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1 .|
Notch-2 is a 300 kDa type I transmembrane glycoprotein that is one of four Notch homologues involved in development (1‑3). Although Notch proteins are structurally and functionally similar, deletion of either Notch-1 or Notch-2 is lethal, demonstrating that not all functions overlap (4, 5). Mice with hypomorphic Notch-2 show defects in development of kidney, heart and eye (6). Notch-2 is upregulated in mature B cells and is critical for differentiation to splenic marginal zone B cells (7). Notch-2 is also preferentially expressed in choroid plexus epithelia and neuronal precursors (8, 9). Mouse Notch-2 is synthesized as a 2470 amino acid (aa) precursor that contains a 25 aa signal sequence, a 1652 aa extracellular domain (ECD), a 21 aa transmembrane (TM) segment, and a 772 aa cytoplasmic domain. The ECD contains 35 EGF‑like repeats and three Lin-12/Notch repeats, while the cytoplasmic region shows six ankyrin repeats, a glutamine‑rich domain and a PEST sequence. Binding of ligands, including Jagged and Delta-like molecules, has been localized to the 11th and 12th EGF-like repeats of human Notch-1 (corresponding to aa 413‑492 in mouse Notch-2) (10). Notch receptors undergo post‑translational furin-type proteolytic cleavage (11). This forms a heterodimer through the interaction of a hydrophobic area in the ligand‑binding extracellular region with the TM/cytoplasmic portion (11, 12). Upon ligand binding, additional sequential proteolysis by
TNF‑converting enzyme (ADAM‑17) 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 (13). Mouse Notch-2 ECD (aa 26‑528) shares 93%, 96%, 92% and 92% aa identity with the corresponding regions of human, rat, canine, and bovine Notch-2, respectively. This region also exhibits 55‑58% aa identity with mouse Notch-1 and Notch-3.