Figure 1: Norrin induces endocytosis of the FZD4 receptor and associated membrane proteins. | Nature Communications

Figure 1: Norrin induces endocytosis of the FZD4 receptor and associated membrane proteins.

From: Norrin-induced Frizzled4 endocytosis and endo-lysosomal trafficking control retinal angiogenesis and barrier function

Figure 1

(a) HeLa cells incubated with AP-Norrin-mycHis conditioned medium on ice were washed and then subjected to a 30 min 37 °C stimulus to induce endocytosis. Staining reveals both cell surface bound and internalized Norrin (via myc). Inset: transfection of co-receptor LRP5 and co-activator TSPAN12 is not sufficient to mediate binding or internalization of Norrin. (b) Removing surface bound Norrin by an acid wash step reveals specifically internalized Norrin. (c) FZD4 shows predominantly cell surface localization in the absence of Norrin. (d) Norrin-induced FZD4 internalization in a cell stained after permeabilization. (e) V5-FZD4 at the cell surface of intact cells was labelled with a V5 antibody. Induction of endocytosis with Norrin and subsequent acid wash allows to specifically reveal internalized FZD4 via the co-internalized V5 antibody (‘antibody feeding’). (f–h). Norrin and FZD4 co-localize in endocytic puncta. Co-activator TSPAN12 and co-receptor LRP5 are co-internalized. Boxed areas in f–h are shown enlarged on the right with the DAPI channel omitted. Internalized FZD4 is specifically revealed by V5 antibody feeding. A similar experiment without V5 antibody feeding is shown in Supplementary Fig. 1a. Scale bars, 10 μm.

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