Figure 2.

PK-resistance assay of in vitro generated GSS-associated misfolded rec-PrPs. Western blot representing the spontaneously generated, misfolded, human rec-PrP after PK-digestion (A) and human (wild type and mutated) rec-PrPs based on PMCA substrates (B). Brain homogenates from three human TSE affected patients are included as controls: GSS P102 129V, GSS A117V 129V and sCJD MM1. Samples were digested with 12, 25 and 85 μg/ml of PK, respectively. The PK digestion (25 μg/ml) of the misfolded recombinant samples show a band of approximately 17 kDa corresponding to the PK-resistant 90-230 fragment of the protein (→). The recPMCA round from where each seed was scaled up: P102L 129M rPrP^res round 24, P102L 129V rPrP^res round 25, A117V 129M rPrP^res round 16, A117V 129V rPrP^res round 21, P105L 129M rPrP^res round 30, P105L 129V rPrP^res round 30, D178N 129V rPrP^res round 30 and V210I 129M rPrP^res round 30. Membranes were developed with 3F4 monoclonal antibody (1:10,000). Mw: Molecular weight. Despite all the samples shown were run in the same gel, the blot was cropped as indicated by the vertical grey line to avoid displaying unrelated samples run between the ones shown.