Figure 2

MoPrP105-132 avoids early endosomes and lysosomes. Neuroblastoma incubated with biotinylated MoPrP105-132 at 37°C, then fixed and stained with Texas Red-streptavidin (red) and TfR-FITC (green) or LAMP-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) was not observed between MoPrP105-132 and TfR or (B) between MoPrP105-132 and LAMP-1. (C) Co-localisation of biotinylated scrambled MoPrP105-132 (red) with LAMP-1 (green) was evident. Neuroblastoma cells were incubated with biotinylated MoPrP105-132 (red) for 90 minutes, then fixed and stained with GM130 (green) or Grp78 (green). (D) Co-localisation was observed between MoPrP105-132 and GM130 and (E) between MoPrP105-132 and Grp78. Scale bars, 5 μm. (F) Separation of endosomal compartments. Neuroblastoma cells were pulsed with iron dextran beads, before incubation with MoPrP105-132-FITC for 1 hour. Lysosomes (F3) were extracted from whole microsome extracts (MEx) using a magnetic column and early endosomes (F1) and intermediate fraction (F2) isolated using a density gradient. Western blot analysis revealed MoPrP105-132 was present in TfR negative and LAMP-1 negative fractions (F2).