Fig. 7. Disruption of rafts or displacement of PrP from rafts promotes the endocytosis of PrP in the absence of Cu²⁺. (A) SH-SY5Y cells expressing PrP^C were surface biotinylated and either untreated or treated with 1 mM MßCD in the absence or presence of 500 µM tyrphostin A23. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrP^C. Cells were then lysed, and PrP^C was immunoprecipitated from the sample using antibody 3F4 and then subjected to western blot analysis. The biotin-labelled PrP^C fraction was detected with peroxidase-conjugated streptavidin. The same samples were also immunoprecipitated using an anti-transferrin receptor antibody. (B) SH-SY5Y cells stably expressing PrP^C or PrP-CTM were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C and then washed three times in PBS. Where indicated, cells were incubated either at 4°C for 10 minutes with PBS containing 1% Triton X-100, or with 1 mM MßCD at 37°C for 45 minutes, prior to fixation. Bar, 10 µm. (C) SH-SY5Y cells expressing PrP-CTM were surface biotinylated and either untreated or treated with 500 µM tyrphostin A23. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrP. Cells were lysed, and PrP-CTM immunoprecipitated and analysed as described in (A). Results are the mean±s.e.m. of three separate experiments done in duplicate each time (n=6), with values of P<0.05 taken as statistically significant where indicated.