Notch localization compared with lipid raft-like microdomains on the endosome surface in S2 cells. (A–C) Colocalization of lipid rafts markers and Notch endocytosed for 60 min at 25°C in Su(dx)-overexpressing S2 cells. (A) EGFP-GPI (green), human Cav1-mRFP (red), and internalized Notch^ECD antibody (blue). (B) EYFP-Rab7 (green), Cav1-mRFP (red), and Notch^ECD antibody (blue) at 25°C. Note that Cav1 is mainly found inside the endosome (arrow), but there is also a spot on the limiting membrane (arrowhead). (C) EYFP-Rab7 (green), Cav1-mRFP (red), and Notch^ECD antibody (blue) at 18°C. Arrowhead marks Notch colocalization with Cav1. (D) EYFP-Rab7 (green), Cav1-mRFP (red), and Notch^ECD antibody (blue) endocytosed for 60 min at 25°C in Dx-overexpressing S2 cells. Notch (arrow) and Cav1 (arrowhead) are separated into different microdomains on the endosomal surface. Note that without Dx or Su(dx) expression, we were unable to image Notch the limiting endosomal membrane in S2 cells because of the constitutive transfer of Notch into endosomal lumen and more limited endocytic uptake (Shimizu et al., 2014). (E and F) Time-lapse images of Notch-EGFP (green), Cav1-mRFP (red), and SNAP-2xFYVE (blue), expressed in control (E), and Dx-expressing (F) S2R+ cells. (E) Notch is localized to and moves with Cav1-positive (raft-type) membrane domain on endosomal surface (arrowheads) in control cells. (F) In Dx-expressing cells, Notch localizes to discrete patches on the endosome membrane (arrowheads), which are separate from the Cav1-positive membrane domain (arrows).