Immunoblot detection of PrP in brain tissues from tg66 mice. Mice were intracerebrally injected with brain homogenates from human GSS patients expressing PrP mutants Y226X, Q227X and G131V. Mice are identified by their days post-injection (dpi), and can be cross-checked by this identifier in the Tables. Prior to analysis, all samples were concentrated using a PTA procedure as described in the methods, except for panel b, lane 1 (vCJD), which was processed by the standard protocol . Panel a shows one control uninfected mouse (C) and three tg66 mice infected with brain from the patient with mutant Y226X. Samples in lanes 1–4 had no PK treatment (−), and samples in lanes 5–8 were treated with PK at a concentration of 20 μg/ml (+). Following PK digestion, two Y226X injected mice (593 and 601) showed faint PK resistant PrPSc bands at 29, 24 and 19kD (lanes 5 and 7) as indicated on right margin. Samples in lanes 1–4, which were not PK-treated, showed similar PrP bands in all the mice. Since this PrP pattern was also found in the uninfected control, this would appear to be PTA-precipitated aggregated PK-sensitive PrPC, which was present in all tg66 mice. In panel b, two additional Y226X injected mice (718 and 716) in lanes 2 & 3 had a strong PrPSc signal. In addition, three Q227X injected mice (lanes 4–6) and eight G131V- injected mice (lanes 7–15) had no detectable PrPSc. Mouse 650 was analyzed both without and with PK treatment in lanes 9 and 10. The blot in panel a was probed with a combination of monoclonal anti-PrP antibodies 3F4 and SAF32, and panel b was probed with antibody 3F4 alone. Approximate molecule weights calculated for each PK-resistant band are shown in margins