Fig. 5. Effective suramin analogues induce aggregation of PrP^c independently of the mode of surface anchorage. (A,B,C) N2a cells were transiently transfected with 3F4-tagged wtPrP, CD4-PrP or Thy-1 and treated with 200 µg/ml of the indicated compounds (10 or 200 µg/ml for NN) for 3 days. Postnuclear lysates were subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl. Pellets and supernatants were run on SDS-PAGE and analysed by immunoblotting using the monoclonal antibody 3F4 or monoclonal anti-HA antibody to detect Thy-1. With the exception of ANTS and NN, Suramin and all the tested derivatives induce formation of insoluble PrP-aggregates (A, lanes 3,5,7,9,11,13) whereas PrP^c remains soluble in cells treated with ANTS (A, lane 16) or NN (A, lanes 22 and 24). CD4-PrP also partitioned in the insoluble fraction upon incubation of cells with suramin derivatives (B, lanes 3, 5, 7, 9, 11), whereas the same treatment had no effect on Thy-1 solubility (C).