Additional file 4 of Ubiquitylation is required for the incorporation of the Notch receptor into intraluminal vesicles to prevent prolonged and ligand-independent activation of the pathway

Additional file 4: Figure S4. Impact of Su(dx) on the endocytosis and activation of endogenous N. Su(dx) was constitutively expressed under control of hh-GAL4 in the posterior compartment of third instar larvae wing imaginal discs. Expression was performed at 29°C. The localisation of endogenous N was revealed by anti-NECD antibody staining, the activity of the pathway by expression of NRE-GFP. (A) Expression of Su(dx) caused the formation of a small scar at the expression domain boundary (arrow) and a suppression of the expression of the activity reporter NRE-GFP (arrow), indicating that it suppresses the activity of the pathway. (C) Irrespective of Su(dx) over-expression, endogenous N clearly localised at the apical plasma membrane, indicated by the honeycomb pattern (arrows in the upper panels). This result indicates that the constitutive over-expression of Su(dx) had no obvious impact on endocytosis of endogenous N. Upper panels show a Z- projection of the A/B axis. The apical plasma membrane is indicated by arrows. The dashed yellow line highlights the expression boundary. Lower panels show a focal plane of the apical plasma membrane. Due to the scar formation of the wing imaginal discs, the apical plasma membrane of the anterior and posterior compartment is slightly shifted in its focal plane. Therefore, (C) shows the focal plane of the apical plasma membrane of the anterior control compartment, while (C’) shows the apical domain of the posterior Su(dx) expressing compartment. (D) Dx over-expression resulted in a formation of enlarged N positive vesicles (magnification in insert). (B) In contrast, Su(dx) had no obvious impact on endogenous N vesicle formation.