Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Neur^G167E exhibits cytoplasmic localization in S2 cells and in embryonic neural tissue. Localization of V5-tagged Neur proteins is shown in S2 cells (panels A and B) and in embryonic neuroectoderm (C and D). Neur^PA (A) and Neur^G167E (B) are shown in red and nuclei are stained with DAPI, shown in blue (A' and B'). Overlays are shown in A" and B". Similar to in vivo, V5-Neur^PA exhibits predominantly plasma membrane localization (A, arrow) while Neur^G167E is mostly present in cytoplasmic puncta (B). In embryos, Neur^PA is localized to the plasma membrane as well as cytoplasmic puncta (C). In contrast, Neur^G167E exhibits predominantly cytoplasmic staining (D).
• Supplemental Figure 2 - The G167E mutation increases Neur localization to HRS-containing endosomes in S2 cells. (Panels A and B) Staining for V5-tagged Neur^PA (A) or Neur^G167E (B) is shown in red, Hrs staining is shown in green (A’ and B’). Overlays are shown in A” and B” and co-localization is shown in yellow. V5-Neur^PA is localized to the plasma membrane and is present in some cytoplasmic puncta (A). Some of these puncta co-localize with Hrs (A", arrow). V5-Neur^G167E is present in many more cytoplasmic puncta compared to wild-type (B) and exhibits increased co-localization with Hrs (B"). (C) Quantification of co-localization between Hrs and Neur^PA or Neur^G167E in S2 cells. Neur^PA co-localizes with Hrs in ~20% of cells. In contrast, Neur^G167E co-localizes with Hrs in >50% of cells. This increase is statistically significant when compared to wild-type. For each protein three separate transfections were analyzed (n=3), each with a sample size of 40. Error bars represent standard error. The asterisk indicates statistical significance (*p<0.05).