Figure 1.

DSL ligands induce clustering and transendocytosis of Notch1. (A) Coculture and staining protocol for Dll1 endocytosis and N1 transendocytosis (see Materials and methods for details). Small filled circles represent intracellular vesicles. (B and C) HA-N1 C2C12 cells were cocultured with Dll1 (B) or parental L (C) cells in the presence of rabbit anti-Dll1 extracellular domain (ECD) antibodies to track Dll1 internalization. Cells were then fixed, permeabilized, stained with an HA antibody (16B12) conjugated to Alexa Fluor 488 to detect N1 N terminus (green) and anti–rabbit Alexa Fluor 633 to detect Dll1 antibodies (red), and imaged by confocal and differential interference contrast (DIC) microscopy. Arrows indicate N1 puncta polarized at interfaces of Dll1–N1 cells; arrowheads indicate colocalization of N1 and Dll1 within the Dll1 cell (yellow). Boxes indicate enlarged regions. Overlays are composites of fluorescent and DIC images. Images were uniformly adjusted using the levels function in Photoshop (Adobe). Bar, 5 μm.