Mutant PrPs acquire abnormal biochemical properties in PC12 Tet-on cells.

(A) Cells transfected with WT, M128V, D177N/M128, D177N/V128 or PG14 PrP were induced with 1 µg/ml dox for 24 h before lysis. 100 µg of protein extract was ultracentrifuged for 40 min at 186,000 x g to separate the soluble (S) and insoluble (P) protein fractions, and PrP was analyzed by Western blot using antibody 3F4. (B) Cells transfected with WT or mutant PrPs were induced with 1 µg/ml dox for 24 h before lysis. 300 µg of protein extract was digested with the indicated concentrations of PK and analyzed by Western blot using antibody 3F4. The bracket indicates the bands corresponding to PK-resistant PrP.