FIG 7.

Alternative substitutions at positions 108 and 202 of rabbit rec-PrP show the relevance of the presence and absence of branched-chain amino acids, respectively, to alterations of misfolding proneness. The histogram shows rec-PrP^res generation over the course of serial in vitro propagation of rabbit recombinant mutated PrPs with alternative substitutions, using recombinant RML and brain-derived RML as seeds. Generation of rec-PrP^res was evaluated by PK digestion and Western blotting (with monoclonal antibody SAF84) for each recPMCA round (rounds 1 to 20). The percentages of tubes showing PK-resistant misfolded rec-PrP are indicated in grayscale according to the legend. For the M108I substitution, both recRML and brainRML were able to induce misfolding. Rabbit rec-PrP with the alternative substitution I202M was not misfolded by brainRML, and the rec-PrP^res induced by recRML was generated in later rounds than those for rabbit I202V rec-PrP.