Fig. 1. PrPT182A acquires scrapie-like characteristics in transfected FRT cells. (A) PIPLC assay. FRT cells were grown on 60 mm dishes and lysed in Triton X-114 buffer. Aqueous phases were accurately recuperated and TCA precipitated, while detergent phases were incubated for 1 hour at 37°C in the presence (+) or absence (–) of PIPLC (5U/sample). 2% Triton X-114 was added and the samples were incubated for 10 minutes on ice. Separation was repeated and detergent (D) and aqueous (A) phases were recovered separately, immunoprecipitated and revealed by western blot. D, diglycosylated PrP; M, monoglycosylated PrP; U, unglycosylated PrP. (B) Triton/DOC insolubility assay. After lysis in Triton/DOC buffer, lysates of FRT cells were ultracentrifuged to separate detergent-soluble (S) and detergent-insoluble (P) molecules. The proteins were TCA precipitated and PrPs were separated by SDS-PAGE and analysed by western blot. (C) Proteinase K (PK) digestion assay. FRT cells were lysed in Triton/DOC buffer in the absence of protease inhibitors, and treated where indicated (+) with PK (3.3 µg/mg of protein) at 37°C for 2-10 minutes. The proteins were then TCA precipitated, separated by SDS-PAGE and immunoblotted. Note that the ratio between the amount of total proteins loaded in PK-untreated/PK-treated samples is 1:3.