Fig. 3. Suramin induces intracellular re-routing of PrP from post-ER compartments to acidic vesicles. (A) PrP is not detectable by surface FACS analysis in Suramin-treated cells. The left panel shows the surface FACS analysis of 3F4-N2a cells [the bold line is PrP reactivity without Suramin; the broken line is in the presence of Suramin (200 µg/ml for 24 h)]. The y-axis denotes the number of cells, the x-axis the fluorescence intensity (lg-scale). The polyclonal rabbit anti-PrP antibody A4 was used. The right panel shows the identical analysis with permeabilized cells (intracellular FACS). (B) No release of surface PrP by PIPLC in cells treated with Suramin. 3F4-N2a cells were incubated for 3 days with Suramin (200 µg/ml medium), treated with PIPLC, and the cellular lysate preparations analyzed in immunoblot (3F4). In non-Suramin-treated cells, PIPLC treatment results in the expected reduction of PrP in the cellular lysate fraction (lane 2); in the presence of Suramin, PIPLC treatment has no effect (lane 4). (C) Suramin induces intracellular retention of PrP in the Golgi/TGN and re-routing to acidic compartments. 3F4-N2a cells were mock treated (panel a) or incubated in the presence of Suramin (100µg/ml for 24 h) [panels (b) and (c)] and analyzed in indirect immunofluorescence experiments using confocal microscopy. (Panel a) PrP is excluded from the cell surface of Suramin-treated cells. Cell surface proteins of fixed and non-permeabilized 3F4-N2a cells were unspecifically surface-labeled with biotin; at the same time PrP present at the cell surface was specifically labeled with the antibody 3F4. The left panel shows surface biotinylated proteins (biotin), the middle panel cell surface PrP (3F4), and the right panel the overlay of both signals in yellowish color (merge). The upper right cell does not express detectable levels of 3F4-PrP, which indicates the specificity of the PrP antibody used. (Panel b) Suramin induces the retention of PrP in a Golgi/TGN compartment. 3F4-N2a cells were treated with Suramin, fixed, permeabilized and incubated with a polyclonal anti-mannosidase II antiserum to localize the medial to trans-Golgi compartments (left panel, manII). In parallel, PrP was analyzed with 3F4 (middle panel). In the right panel the overlay of both signals indicates co-localization (merge). (Panel c) PrP is re-routed to acidic compartments in Suramin-treated cells. Cells and treatment as in panel (b), with the exception that leupeptin was added. To stain for acidic endosomal/lysosomal compartments, an antibody against the acidotropic amine DAMP was used. The left panel shows the DAMP staining, the middle panel is PrP (3F4), and the right panel denotes the overlay of both signals (merge).