Conversion activity of wild-type and mutant PrP<sup>C</sup> 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<sup>C</sup> 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<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
