Mouse DNER Antibody

DNER in Mouse Brain.

DNER was detected in perfusion fixed frozen sections of mouse brain (cerebellum) using Goat Anti-Mouse DNER Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2254) at 5 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to Purkinje neurons and molecular layer. View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Detection of Human DNER by Immunocytochemistry/Immunofluorescence

DNER does not activate, function with, or bind to Notch, but known Notch ligand DLL1 does.(A) Pooled luciferase results from 4 separate experiments (normalized to the mean of empty vector in each experiment). U2OS cells were transfected with ligand (DLL1, DNER, or EV), and separately a population of U2OS cells was transfected to express Notch, the control luciferase Renilla, and TP1, a promoter that expresses firefly luciferase when Notch is activated. The two populations were co-cultured 24 hours after transfection (trans configuration), and activity read after an additional 24–48 hours of incubation. (B) C2C12 cells (myoblasts) were incubated with differentiation media (2% horse serum) that either had pre-clustered DLL1-fc (1:1), pre-clustered DNER-fc (1:1), un-clustered DNER-fc, or fc only, all at a ratio of 1:150 in media. Cells were incubated for 72 hours, then fixed, and stained for the presence of myosin heavy chain (MHC) and nuclei. By measuring the percent of total nuclei that were inside of differentiated MHC positive myotubes, fusion indexes were calculated. (C) DNER (top left, green) transfected U2OS cells were not labeled by pre-clustered Notch-fc (top middle, red) but DLL1 (bottom left, green) transfected U2OS cells were labeled by pre-clustered Notch-fc (bottom middle, red). Merged images are shown at far right. Scale 10 μM. **** = p value <0.0001. *** = p value 0.002. ns = not significant. DLL1 = Delta-like 1, a known Notch Ligand, DNER = Delta/Notch-like epidermal growth factor (EGF) related receptor, GSI = gamma-secretase inhibitor, fc only = rabbit anti-human-fc. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0161157), licensed under a CC-BY license. Not internally tested by R&D Systems.