Change in Notch distribution on the endosomal limiting membrane after ESCRT-III knock-down. (A–D) Microdomains of the limiting membrane were labeled with Cav1-EGFP (green in A and C) or EGFP-clathrin light chain (green in B and D) together with Notch^ECD (red), and mCherry-2xFYVE, an endosomal membrane marker (blue), expressed in S2R+ cells. (A′–D′) Fluorescence intensity of Cav1 or CLC (green) and Notch (red) were measured and plotted along the limiting membrane of each endosome (curved arrows in A–D). (E and F) Quantification of Pearson’s correlation coefficient. (E) Notch/Cav1 colocalization in control S2R+ and Tsg101 KD cells and reduced (or no) correlation in Shrub KD and Dx/VPS4^EQ expressing cells. (F) Independent distribution of Notch and clathrin light chain in control S2R+ and Tsg101 KD cells and correlative localization in Shrub KD and Dx/VPS4^EQ expressing cells. (G–M) Downregulation of ESCRT-III function alters Notch localization on the endosome membrane. (G–L) Images of Notch^ECD antibody (green) endocytosis-uptake assay at indicated chase times in control (G and H), Tsg101 KD (I), Shrub KD (J and K), and VPS4^EQ expressing (L) S2 cells, Cav1-mRFP (purple). (M) Time course of colocalization between Notch^ECD antibody uptake and Cav-1-mRFP showing percentage of Notch^ECD on Cav1-mRFP positive spots, scored in control, Tsg101 KD, Shrub KD, and VPS4^EQ overexpressing cells. Error bars in E, F, and M are SEM. In E and F, sample sizes are indicated in the figure. *, ** indicate P < 0.05 and 0.01, respectively, by two-tailed Student’s t test. In M, data is from three experimental repeats with 50–100 puncta scored per repeat; error bars represent SEM.