Figure 2. Endocytosis of PrP^C is blocked by siRNA directed against LRP-1, but not LRP-1B.

SH-SY5Y cells expressing PrP^C were incubated with a 2 μM solution of the indicated LRP siRNA prepared with DharmaFECT-1 transfection reagent in OptiMEM for 30 h. (A) Cell lysates were immunoblotted for LRP1 expression using Ab 5A6 and LRP1B expression using a goat polyclonal antibody, along with PrP^C and β-actin. (B) Cells were surface biotinylated and treated with or without 100 μM Cu²⁺ for 20 min at 37 °C. Prior to lysis, the cells were, where indicated, incubated with trypsin to digest cell-surface PrP^C. Cells were then lysed and total PrP^C immunoprecipitated from the sample using antibody 3F4. Samples were then subjected to Western blot analysis. The biotin-labelled PrP^C fraction was detected with HRP-conjugated streptavidin. (C) Densitometric analysis (means±S.E.M.) for multiple blots (lanes 5 and 6 in B) from three separate experiments. *, Students t-test with values of P<0.05 were taken as statistically significant.