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.

<p>(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&lt;0.01, ***p&lt;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.</p

Conversion activity of brain derived PrP^C in the CAA seeded with infected brain homogenates.

<p>(A) UBH from balb/c (WT) mice were subjected to the CAA in the presence of IBH for differing periods of time (0–24 hours). B) The CAA was performed for 16 hours using IBH added to DPBS, or UBH from KO, WT or PrP over expressing Tga20 (TG) mice. DPBS represents total (DPBS⁻ without PK treatment), and protease resistant (DPBS⁺ with PK treatment) PrP present in the IBH used to seed the CAA. Relative PrP^C expression (without PK treatment) is shown in right of panel for KO, WT and TG mice. Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight (or as indicated) incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Blots developed with 03R19. Molecular weights (kDa) are shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism).</p

Conversion activity of wild-type and mutant PrP^C expressed in RK-13 cells in the CAA following chlorate treatment to modify the sulphation of GAG.

<p>The CAA was performed using lysates prepared from RK-13 cells expressing WT (101P) and mutant (101L) moPrP. A) Quantification of conversion activity of 101P and 101L moPrP left untreated (−) or treated with 30mM chlorate (+) or UBH (Brain). B) Western blot analysis of PrP^C expression in 101P and 101L moPrP left untreated (−) or treated with 30mM chlorate (+). Equivalent protein loaded in each lane, blots probed with beta-tubulin. CAA performed using (C) 101P-moPrP and (D) 101L-moPrP cells left untreated (−) or treated with 30mM chlorate (+). Conversion activity was determined as the fold increase in immunoreactive signal relative to puroRK reactions after overnight incubation at 37°C and treatment with PK (100µg/ml, 1hour at 37°C). Blots developed with 03R19. Molecular weight (kDa) is shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p&lt;0.05 two-tailed t-test of indicated pairs. In (C) and (D) CAA performed using KO and WT mouse brain homogenates (with quantitation shown as brain in A) and cell lysate derived from puroRK (N), 101P (P) and 101L (L) moPrP expressing cell lines. Truncated fragment (←) was not a consistently observed in either wildtype or mutant cell lines and was not included in analysis.</p

Binding of wild-type and mutant PrP^C expressed in RK-13 to sGAG.

<p>A) The ability of wildtype (101P) and mutant (101L) moPrP expressed in RK-13 cells to bind heparin sepharose beads in the presence of increasing concentrations of NaCl (50, 100, 125, 300, 1000mM), was determined by western blot analysis. N shows neat input. B) PrP^C bound to heparin sepharose in the presence of 50mM NaCl was treated with PNGaseF before western blot analysis. Full length (F) and truncated (T) PrP species are shown. C) The percentage of 101P (dotted line) and 101L (solid line) moPrP bound to heparin sepharose in increasing concentrations of NaCl was determined relative to binding in 50mM NaCl. Blots developed with 03R19. Molecular weight (kDa) is shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. Binding differed significantly by Two-way ANOVA (p&lt;0.001).</p

Conversion activity in the CAA following Benzonase treatment of UBH or IBH.

<p>A) The CAA was performed in the presence of the indicated concentrations of MgCl₂ and B) performed following pre-treatment as indicated. Conversion activity was determined as the fold increase in immunoreactive signal of treated samples relative to their equivalent KO reactions after overnight incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Quantification is based on at least three experiments, mean and SEM are shown. **p&lt;0.001 or *p&lt;0.05 using one-way analysis of variance (ANOVA) with Dunnet's test for multiple comparisons against the indicated control (GraphPad, Prism).</p