Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor
Figure 4
Inhibition of hamster brain PrPC conversion by dominant negative mutant PrP.
Reactions containing either purified brain-derived HaPrPC alone (−Mutant, lanes 2–5) or in combination with either Q172R, immunopurified Q172R (Q172R Pure), T215K, or different concentrations of Q219K HaPrP mutant substrates (+Mutant, lanes 8–11), as indicated, were subjected to three rounds of serial propagation. Q172R Pure was tested at ∼1∶2 (Mut∶WT) ratio; Q172R Low was tested at ∼1∶5 ratio; and all other conditions were tested at ∼5∶1 ratio. In each blot, an arrowhead demarks the expected mobility of the ∼27–30 kDa PK-resistant brain-derived PrPSc species. All reactions were supplemented with synthetic poly(A) RNA. In all blots, a sample containing wild type or mutant HaPrP substrate not subjected to proteinase K digestion is shown in the lane(s) preceding the corresponding PK-digested samples as a reference for comparison of electrophoretic mobility (−PK WT or Mut). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K for 1 hr at 37°C (+PK).