Fig. 8. Suramin derivatives, in contrast to suramin, do not exert their aggregation effect on lysosome-localised LAMP-PrP. (A) N2a cells transiently transfected with wild-type PrP or PrP-LAMP were treated for 48 hours with 200 µg/ml NF449, NF023 or suramin. Cells were then lysed and subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl. Supernatants (S) and pellets (P) were analysed by immunoblot analysis using the monoclonal antibody 3F4. Suramin (lane 11) but not NF449 or NF023 (lanes 7,9) significantly affected the solubility of lysosome-localised LAMP-PrP, confirming a different site of action of the tested compounds. (B) 3F4-N2a cells were biotinylated for 15 minutes on ice. Suramin, NF449 or NF023 were added to the culture medium and cells were placed at 37°C for 4 hours. Cells were then lysed and subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl. Supernatants (S) and pellets (P) were precipitated with the polyclonal antibody A7, analysed by immunoblotting, and biotinylated proteins were detected with streptavidin. Although surface-localised PrP^c is soluble in mock-treated cells (lane 1), all tested compounds affect the solubility of this PrP^c population (lanes 3,5 and 7).