Deltex-induced colocalization of Notch and ESCRT-0 complex on a microdomain of the endosomal limiting membrane. (A–D) Localization of Notch^ECD antibody endocytosed for 60 min (green) in S2 cells expressing Venus-Dx compared with (A) Venus-Dx (red) and mRFP-Rab7 (blue), (B) EGFP-clathrin heavy chain (red) and mRFP-Rab7 (blue), (C) mRFP-clathrin light chain (red) and EYFP-Rab7 (blue), (D) expressed STAM-HA (red) and EYFP-Rab7 (blue). Arrowheads mark sites of colocalization. (E) In S2 cells overexpressing Su(dx)-V5 and mRFP-clathrin heavy chain, Notch (arrowhead) localizes to a discrete patch outside of the clathrin-marked subdomain (arrows). (F and G) Fluorescence intensity of Cav1 or CLC (green) and Notch (red) were measured around the limiting membrane of each endosome and Pearson’s correlation coefficient of colocalization between internalized Notch and markers for endosomal microdomains determined, including Stam-HA, Hrs-HA, Atg18-HA, GFP-myc-2xFYVE, in Dx-expressing S2 cells and GFP-Actin5C in Dx-expressing S2-R+ cells (F), and mRFP-clathrin heavy chain, EGFP-clathrin light chain, endogenous Hrs, and mRFP-Caveolin in Dx- or Su(dx)-expressing S2 cells (G). Error bars, SEM, no. of samples indicated in the figure.