Notch degradation occurs in the lysosomes.

The anti-HA antibody uptake assay was performed as in figure 2. After 90 minutes of chase, cells were fixed, permeabilized and incubated with an anti-LAMP-1 antibody, followed by a mouse-adsorbed rabbit anti-rat and then Al555-coupled anti-rabbit antibodies. Insets represent enlargements (fourfold) of the boxed regions. The photographs are representative of a large number of observed fields. The relative amount of Notch-containing vesicles also positive for LAMP-1 was calculated from 10 cells in each condition. In average less than 25% of Notch vesicles were LAMP-1 negative in MD-FL+Leupeptin, whereas less than 25% of Notch vesicles were positive for LAMP-1 in ID-FL cells (+/− Leupeptin).