Figure 4. Cu²⁺-stimulated endocytosis of PrP^C is unaltered either by overexpression or knockdown of APP.

(A) SH-SY5Y cells expressing PrP^C were stably transfected with a pIREShyg vector containing the cDNA encoding APP₇₅₁. Mock transfectants were created by transfection of an empty pIREShyg vector. Lysates were immunoblotted for APP, PrP^C and β-actin. (B) Cells stably expressing APP₇₅₁ were surface biotinylated and incubated with or without 100 μM Cu²⁺ for 20 min at 37 °C. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrP^C. Cells were then lysed and total PrP^C 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. (C) SH-SY5Y cells expressing PrP^C were incubated for 48 h with a 2 μM solution of the siRNA against APP, prepared with DharmaFECT-1 transfection reagent in OptiMEM. Mock-transfectants were incubated in the presence of DharmaFECT-1 only. Lysates were immunoblotted for APP, PrP^C and β-actin. (D) Endocytosis experiment as described in (B) using the cells incubated in the presence or absence of APP siRNA. (E) Densitometric analysis (means±S.E.M.) for multiple blots from three separate experiments performed in (B) and (D).