Fig. 3.

Cells with Rings and Dots contain PrD-GFP in the same prion conformation. (A Upper) Crude lysates from cells displaying diffuse, Ring or Dot fluorescence were adjusted to equal protein concentrations and serial dilutions (1:2 steps) were loaded onto an SDS gel and analyzed by SDS/PAGE and Western blotting with an anti-GFP antibody to reveal the amounts of PrD-GFP in the different lysates. (A Lower Left) The three different lysates were analyzed by semidenaturing agarose gel analysis (SDD-AGE) followed by Western blotting with an anti-GFP antibody. Lysates from cells with Rings and Dots, but not diffuse PrD-GFP, contain SDS-resistant high-molecular-weight aggregates of PrD-GFP. (A Lower Right) Purified PrD-His was seeded with the three different lysates in vitro and analyzed by SDD-AGE and Western blotting with an anti-His-tag antibody. SDS-resistant high-molecular-weight aggregates were formed with comparable kinetics. (B) Protein transformations of a [psi^-] tester strain (red colony color) were performed with crude lysates from cells with diffuse PrD-GFP fluorescence or Rings and Dots. The prion status and strain of the transformants was determined by colony color. Both types of aggregates induced a strong [PSI⁺] strain (light pink color) with similar efficiencies.