Fig. 5. Effect of Cu²⁺ on the distribution of PrP in detergent-insoluble rafts. SH-SY5Y cells expressing PrP^C, PrP-N or PrP-oct were surface biotinylated and incubated in the absence or presence of 100 µM Cu²⁺ along with 500 µM tyrphostin A23. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP constructs were immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP^C fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected using anti-flotillin-1 and H68.4 antibodies, respectively.