Figure S3.

Endocytosis through GPI-Protein Positive and Negative Routes in S2 Cells, Related to Figure 3

(A–D) Immunostained cells after Notch antibody uptake endocytosis assay (Notch, purple; GPI-GFP green).

(A) Basal Notch endocytosis of N^D505A is into GPI-GFP positive vesicles.

(B) Dx expression induces endocytosis of N^D505A through GPI-GFP negative vesicles.

(C and D) Basal Notch endocytosis of N^R2027A is into GPI-GFP positive vesicles (C) and this is not altered by Dx expression (D).

(E) Quantification of N localisation as % cells with mainly plasma membrane localisation (PM), vesicular localisation (V) or a mixture of plasma membrane and vesicular (PM=V).

(F) % Notch containing vesicles which are GPI-GFP positive or negative. Data in E,F displayed as mean ± SEM from minimum of 3 repeats, each scoring minimum of 60 cells or vesicles per repeat, ^∗ indicates p<0.05 compared to respective controls (Student t-test).

(G–I) GFP antibody uptake assay of GPI-GFP endocytosis (GFP-total green; GFP antibody, purple).

(G) In control cells at 25^oC, most pulse labelled GFP is endocytosed (arrowheads).

(H and I) GPI-GFP endocytosis is reduced by cholesterol depletion (H) or by lowering the temperature to 18^oC (I).

(J) Quantification of anti-GFP localisation in endocytic uptake assay shown as % cells with mainly plasma membrane GFP localisation (PM), vesicular localisation (V) or a mixture of plasma membrane and vesicular (PM=V). Data displayed as mean ± SEM from minimum of 3 repeats, each scoring minimum of 60 cells per repeat, ^∗ indicates p<0.05 compared to respective controls (Student t-test).

(K and L) Endocytosed GPI-GFP traffics through Rab5 (K) and Rab7 (L) positive vesicles. (Total-GPI-GFP, green; GFP antibody marking endocytosed GFP (blue); Rab5-RFP (K) or Rab7-RFP (L), red). GFP colocalisation with Rab5 or Rab7 indicated with arrowheads.