Figure 2. Conversion activity of brain derived PrP^C in the CAA seeded with infected brain homogenates is sensitive to ionic strength and inhibited by the specific depletion of heparan sulphate.

(A) The CAA was performed using IBH diluted in UBH prepared from WT and KO mice in Tris-HCl pH 7.4 and the indicated concentrations of NaCl. ** Indicates a significant reduction in conversion activity relative to 125mM NaCl. B) The CAA was performed using IBH diluted in UBH prepared from WT mice in 125mM NaCl/Tris-HCl pH 7.4 after treatment with Heparinase I (H), Heparinase III (HS), Chondroitinase ABC (Ch), their corresponding buffers (underlined) or without treatment (Con). Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Quantification (A, B) is based on at least three experiments, mean and SEM are shown. **p<0.01, ***p<0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism). C) The amount of sGAG purified from UBH treated with Heparinase I (H), Heparinase III (HS) and Chondrotinase ABC (Ch) or untreated (Con) was determined by Blyscan analysis and normalised to the amount of sGAG recovered from buffer controls (not shown). D) The absorbance (254nm) of sGAG eluted from a Q-Sepharose HiTrap anion exchange column in increasing concentrations of NaCl (0–1M). GAGs were purified from control (□), Heparinase I treated (⋄) and Heparinase III treated (○) or Chondroitinase ABC treated (+) brain homogenates. Quantification (C, D) is based on an analysis performed in duplicate.