Fig. 2. Aggregated Sup35 Contributes Translation Termination Activity to [PSI⁺] Cells.

a. The levels of soluble Sup35 relative to a wildtype [psi] strain and stop-codon readthrough relative to wild-type [PSI⁺] strain were determined in lysates from wildtype [psi] (gray square), wildtype [PSI⁺] (white triangle) and a [psi] strains expressing Sup35 from a doxycycline-repressible promoter (P_tet; black circles). These strains also expressed a reporter for stop-codon readthrough (GST(UGA)DsRedNLS). The levels of Sup35 and GST were determined by quantitative immunoblotting. Error bars represent STDEV; n= 6. b. The relative levels of soluble (black circles) and aggregated (white squares) Sup35 in a P_tet-SUP35 [PSI⁺] strain were determined at the indicated generations following doxycycline-induced repression of Sup35 synthesis by quantitative immunoblotting. Error bars represent SEM; n=3. c. The relative level of stop-codon readthrough of GST(UGA)DsRedNLS a P_tet-SUP35 [PSI⁺] strain was determined by analysis of lysates by quantitative immunoblotting for GST at various times after addition of doxycycline. Error bars represent SEM; n=3. d. [PSI⁺] (gray) and [psi] (white) zygotes with repressed Sup35 synthesis were analyzed by microscopy to determine the number of generations supported by the existing pool of Sup35 in the absence (-) and presence (+) of guanidine hydrochloride (GdnHCl). Error bars represent SEM; n≥ 20; *p=0.0001 by unpaired Student’s t-test.