Michael Jones , Darren Wight, Rona Barron, Martin Jeffrey, Jean Manson, Christopher Prowse, James W. Ironside, and Mark W. Head
Author affiliations: University of Edinburgh, Edinburgh, Scotland, UK (M. Jones, D. Wight, R. Barron, J. Manson, J.W. Ironside, M.W. Head), Veterinary Laboratory Agency, Edinburgh (M. Jeffrey); Scottish National Blood Transfusion Service, Edinburgh (C. Prowse)
Figure 1. Amplification of PrPd by PMCA from bovine BSE, ovine scrapie, experimental ovine BSE, and human vCJD brain homogenates in substrate homogenates prepared from humanized transgenic mouse brain tissue expressing PrP of each human prion protein gene codon 129 (PRNP-129) genotype. A) Amplification of each PrPd type, as determined by Western blotting using MAb 6H4 to detect PrPres after limited proteinase K digestion, in a PRNP-129MM substrate (top panel, 3-min exposure), a PRNP-129MV substrate (middle panel, 3-min exposure), and a PRNP-129VV substrate (bottom panel, 3-min exposure). B) Amplification of each PrPd type, as determined by Western blotting using MAb 3F4 to detect PrPres derived from human PrP after limited proteinase K digestion, in a PRNP-129MM substrate (top panel, 30-s exposure), a PRNP-129MV substrate (middle panel, 3-min exposure), and a PRNP-129VV substrate (bottom panel, 10-min exposure). Limited proteinase K digestion and Western blotting were conducted out as previously described (11). MAb 6H4 (Prionics, Schlieren-Zurich, Switzerland) and MAb 3F4 (Dako, Ely, Cambridgeshire, UK) were used at a final concentration of 50 ng/mL. PrPd, disease-associated prion protein; PMCA, protein misfolding cyclic amplification; BSE, bovine spongiform encephalopathy; vCJD, variant Creutzfeldt-Jakob disease; MAb, monoclonal antibody; PrPres, protease-resistant prion protein; MM, methionine homozygous; MV, methionine/valine heterozygous; VV, valine homozygous. Values on the left are in kilodaltons.
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