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. 2015 Jul 20;210(2):303–318. doi: 10.1083/jcb.201411001

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© 2015 Gomez-Lamarca et al.

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Figure 7. — Effects on Notch trafficking of combined Rme-8 and ESCRT-0 depletion. (A–B″) Distribution of NICD and Rab7. xy section of discs at the subapical plane (top) and xz cross sections of the same specimens (bottom). Notch accumulates in enlarged Rab7 structures upon Hrs knockdown (A; Hrs-Ri+w-Ri) but not in double knockdown (B; Hrs-Ri+Rme8-Ri). (C and D) TEM images of Hrs-Ri;w-Ri (C) or Hrs-Ri;Rme8-Ri (D) with endosomes colored in yellow. (E) Quantification of endosome perimeters from TEM images (WT n = 83, Hrs-Ri+w-Ri n = 33, and Hrs-Ri+Rme-8Ri n = 42). Boxes indicate 25th to 75th percentiles, whiskers represent maximum and minimum values in the dataset, and lines show the median values. Asterisks indicate significance according to one-way analysis of variance test; **, P = 0.01–0.001; ****, P < 0.0001. (F) Superresolution images of single endosomes from Hrs-Ri;w-Ri (left)– or Hrs-Ri;Rme8-Ri (right)–expressing discs, stained for NICD and Rab7, which appear to localize in the same vesicular compartment in Hrs knockdown (arrowheads, left), but not in the Rme-8-Hrs double knockdown, where Notchlocalizes in long tubules (arrows, right). (G and H) Antibody uptake assay for Notch in cultured wing imaginal discs expressing Hrs-Ri;w-Ri (G) or Hrs-Ri;Rme8-Ri (H).