In vitro generated rPrP-res. (A) One round PMCA consisted of 48 cycles of sonication (0.5 minute) and incubation (29.5 minute). At the end, 1/10 reaction mixture was transferred to fresh substrate mixture to start a new round (20). PMCA products were digested with 25 μg/ml PK. C, undigested recPrP. (B) Serial PK digestion of PMCA products. (C) PMCA product was separated into supernatant(s) and pellet(p) by a 1-hour 100,000g centrifugation at 4°C. T, total input; +PK, 25 μg/ml PK digested. (D) With normal mouse brain homogenate (NMBH) as substrate, PMCA was performed with or without rPrP-res seed. Product was digested with 100 μg/ml PK. C, undigested NMBH. (E) After rPrP-res infection, SN56 cells were lysed, digested with 25 μg/ml PK, and centrifuged. The PK-resistant PrP in the pellet was detected by immunoblot analysis. Numbers indicates cell passages. C1, undigested SN56 cell lysate. C2, pellet of PK-digested, uninfected SN56 cell lysates. In all panels, PrP was detected by immunoblot analysis with POM1 anti-PrP antibody except for the right panel in (C). PK digestion was carried out at 37°C for 30 minutes (A, B, and C) or 1 hour (D and E).

Intracerebral rPrP-res inoculation. (A) After injection, the remaining inocula with or without PK digestion were electrophoresed and PrP was detected by immunoblot analysis with POM1 anti-PrP antibody. The insert is a lighter exposure of undigested inocula. (B) Mice injected with rPrP-res showed indicated signs.

Characterization of rPrP-res caused prion disease. Histological analyses of age-and sex-matched control mice (A, C, and E) and rPrP-res injected mice (B, D, and F). Brain sections were stained by hematoxylin and eosin (A) and (B), an anti-GFAP (glial fibrillary acidic protein) antibody (C) and (D), and SAF84 anti-PrP antibody (E) and (F). Immunohistochemical stains were counterstained with hematoxylin. Scale bar represents 50 μm. (G) Brain homogenates of an rPrP-res inoculated mouse or mice inoculated with control inocula 1–3 were digested at 37°C for 1 hour with indicated concentrations of PK. PrP was detected by immunoblot analysis with M20 anti-PrP antibody. (H) Brain homogenates of mice that received second round transmission or control mice inoculated with inoculum diluent (PBS) were digested by 50 μg/ml PK at 37°C for 1 hour. PrP was detected by immunoblot analysis with POM1 antibody. Number indicates the mouse from which the inocula were prepared.