Fig. 7. Effect of overexpression of PrP^C upon its endocytic and biosynthetic trafficking. (A) The percentage of surface-labelled PrP^C on WT (left panel) and Tg20 neurons (right panel) that has been endocytosed is plotted as a function of their relative level of surface PrP^C (measured as the fluorescence intensity of Alexa-Fluor-594–2S Fab bound per µm³). The correlation coefficient and its significance (p) are shown. To avoid saturation of the camera by the 14-fold brighter fluorescence of Tg20 neurons, excitation intensity was turned down to give an overall fluorescence measured that was approximately comparable for the two sets of neurons. (B,C) tg20 neurons, treated for 90 minutes with siRNA^Con (B) or siRNA^LRP1.1 (C), labelled with 2S Alexa-Fluor-594–Fab (red) and Alexa-Fluor-488–Tf (green) were placed at 37°C for 6 minutes. In the control cell shown, 50% of the labelled PrP^C has been internalised, and 35% in the siRNA^LRP1.1-treated cell. (D,E) Examples of immunolabelling of WT (D) and Tg20 (E) neurons for cell-surface PrP (Alexa-Fluor-488–2S fab; green) and internal PrP^C (Alexa-Fluor-594–SAF83, red). Bars, 5 µm.