Fig. 4. Effect of 4 hours of treatment with siRNA^LRP1 upon PrP^C distribution within neurons, and upon LRP1-dependent endocytosis. (A-D) Neurons pretreated with 250 nM siRNA^Con (A), or with siRNA^LRP1.1 (B), siRNA^LRP1.2 (C) or siRNA^LRP1.3 (D), were surface labelled with Alexa-Fluor-488–2S Fab anti-PrP (green), then fixed and permeabilised for labelling internal PrP^C with Alexa-Fluor-594–SAF83 (red). Quantitation is given in supplementary material Table S4a. (E,F) Neurons treated as above with siRNA^Con (E) or siRNA^LRP1.1 (F), fixed, permeabilised and labelled (both surface and internal) with Alexa-Fluor-488 anti-Thy-1 antibody (green). Quantitation is given in supplementary material Table S3b. (G,H) Neurons treated with siRNA^Con (G) or siRNA^LRP1.1 (H) were prelabelled with Alexa-Fluor-488 ₂M^* (green) and Texas Red-Tf and then placed at 37°C for 2 minutes. (I,J) Neurons treated with siRNA^Con (I) or siRNA^LRP1.1 (J) were prelabelled for PrP^C with Alexa-Fluor-594–Fab (red), and the transferrin receptor with Alexa-Fluor-488–Tf (green), and placed at 37°C for 2 minutes. Bars, 5 µm.