Mutant PrP in the brains of Tg(FFI) mice is insoluble and mildly protease-resistant.

<p>(A, B) The indicated amounts of total proteins extracted from the brains of a C57BL/6J mouse (non-Tg, 360 days old), a Tg(FFI-10^+/-)/<i>Prnp</i>^0/0 (87 days old), a Tg(FFI-15^+/-)/<i>Prnp</i>^0/0 (32 days old) and a Tg(FFI-K5^+/-)/<i>Prnp</i>^0/0 (601 days old) (panel A), and from a Tg<i>a</i>20 (113 days old), a Tg(WT-E1^+/-)/<i>Prnp</i>^0/0 (460 days old), a Tg(FFI-26^+/-)/<i>Prnp</i>^0/0 (227 days old) and a non-Tg mouse (307 days old) (panel B), were analyzed by Western blot with monoclonal antibody 12B2. (C, D) Brain lysates prepared from mice of the following genotypes and ages were ultracentrifuged at 186,000 x g for 40 min, and PrP in the supernatants (S lanes) and pellets (P lanes) was analyzed by Western blotting using the 12B2 antibody: non-Tg, 61 days; Tg(FFI-10^+/-)/<i>Prnp</i>^0/0, 58 days; Tg(FFI-K5^+/-)/<i>Prnp</i>^0/0, 287 days; Tg(WT-E1^+/-)/<i>Prnp</i>^0/0, 276 days; Tg(FFI-26^+/-)/<i>Prnp</i>^0/0, 371 days. (E) Brain lysates from the Tg(WT-E1^+/-)/<i>Prnp</i>^0/0 and Tg(FFI-26^+/-)/<i>Prnp</i>^0/0 mice used in D were incubated with 0–2 μg/ml of PK for 30 min at 37°C, and PrP was visualized by Western blotting using antibody 12B2. The undigested samples (0 μg/ml PK) represent 25 μg of protein, and the other samples 100 μg. (F) Brain lysates from the Tg(FFI-26^+/-)/<i>Prnp</i>^0/0 mouse and a Tg(CJD-66^+/-)/<i>Prnp</i>^0/0 mouse (322 days old) were incubated with 0 or 0.5 μg/ml of PK as in E, followed by incubation with PNGaseF and Western blot analysis with antibody 12B2. The arrowheads indicate the PK-resistant deglycosylated PrP bands. Molecular size markers are given in kDa.