Western blot analysis of skin tissue from CJD and non-CJD patients

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Fig. 1. Western blot analysis of skin tissue from CJD and non-CJD patients. Western blot analysis of skin tissue from CJD and non-CJD patients. (A) Western blot analysis of proteinase K (PK)–resistant misfolded forms of the prion protein (PrPSc) in skin from the U.S. variant Creutzfeldt-Jakob disease (US vCJD) patient. The blot was probed with anti-C antibody against human PrP(220–231) with an exposure time of 5 or 50 min. The skin samples [PK-treated, PrPSc-concentrated fraction (P4) after enrichment by ultracentrifugation] were treated with PK (25 μg/ml) at 37°C for 1 hour, followed by addition of Roche complete protease inhibitor cocktail. A CJD brain homogenate (brain) was used as a positive control. The black arrow refers to the longer exposure time of the blot on the right corresponding to the right lane of the left panel (at normal exposure). (B) Western blot analysis of PrP from the US vCJD patient skin sample treated with PK and peptide N-glycosidase F (PNGase F) and probed with the 3F4 anti-PrP antibody. The CJD brain homogenate was used as a positive control. (C) Western blot analysis of PrP in skin or brain samples from the UK vCJD patient treated with PK or PK plus PNGase F and probed with the anti-PrP antibody 3F4. (D) Comparison of the amounts of PrPSc in the brain between the US vCJD and UK vCJD cases treated with PK. (E) Western blot analysis of PrPSc in skin samples from sCJD patient nos. 1 to 5, vCJD patient no. 6, and non-CJD patient nos. 8 to 17, treated with PK (top) or PK plus PNGase F (bottom). Skin samples from about 60 to 80 mg of skin tissues for each case (~6 mm × 6 mm in area) were used for PrPSc enrichment and loading onto the gel. The vCJD patient no. 6 skin sample was used as a positive control. The black arrows indicate the PK-resistant deglycosylated PrPSc. The results shown in (A) to (E) are representative of three independent experiments. Christina D. Orrú et al., Sci Transl Med 2017;9:eaam7785 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works