Microdomain markers in S2 cells. (A and B) Colocalization of lipid raft markers and Notch endocytosed for 60 min at 25°C in Su(dx) (A) or Su(dx)-V5 (B) overexpressing S2 cells. (A) GFP-GPI (green), Notch^ECD (red) antibody, and Flotillin-2-HA (blue). (B) GFP-GPI (green), myr-RFP (red), and Notch^ECD (blue) antibody. (C) Luciferase assay of Notch signal activation in S2 cells cotransfected with 0, 0.1, 0.3, 1.0, 3.0, or 10 ng pMT-Cav-GFP or pMT-GFP-GPI. GFP-GPI has a greater impact on Notch signal than Cav-GFP expression. Notch-only represents control Notch-expressing cells that were not cotransfected with Dx or Su(dx). (D and E) Localization of Notch^ECD antibody endocytosed in S2 cells for 60 min (green) when coexpressed with Dx, on Fyve-positive endosomes compared to Atg18a (D) and Actin (E). (F and G) Colocalization of Notch^ECD antibody endocytosed for 60 min with Hrs on Rab7-labeled endosomal membrane in S2 cells overexpressing Dx. Overexpressed Hrs-HA (F) or endogenous Hrs (G) show colocalization with Notch on EYFP-Rab7 or mTagBFP2-Rab7-positive late endosomes, respectively. (H) Time course of colocalization between Notch^ECD antibody endocytosis and mRFP-clathrin heavy chain in Dx or Su(dx)-V5 overexpressing cells. Notch and clathrin colocalize only at a later stage of endocytosis in Dx-expressing cells. Error bars represent SEM. Data from three repeats with 50–100 puncta scored per repeat.