FIG 5.
Repeated cycles of dehydration and hydration does not alter strain emergence. Western blotting (A) and migration analysis (B) of PrPSc from PMCAsi reactions. Negative-control PMCA reactions did not amplify PrPSc (lane 1). Positive-control PMCA reaction mixtures seeded with either HY-infected (lane 8) or DY-infected (lane 2) brain homogenate amplified PrPSc that maintained the 21-kDa (lane 15) or 19-kDa (lane 9) strain-specific unglycosylated PrPSc migration pattern, respectively, after 5 rounds of amplification. Positive-control zero wetting and drying cycle PMCAsi reaction mixtures seeded with both HY and DY (lanes 3 to 7) resulted in the emergence HY PrPSc by round 5. Experimental PMCAsi reaction mixtures seeded with both HY and DY after 10 serial rounds of wetting and drying treatment (lanes 10 to 14) resulted in the emergence of HY PrPSc by round 5. Migration of 19-kDa and 21-kDa molecular weight marker is indicated on the left of the Western blot. This experiment was repeated a minimum of 3 times with similar results.