Figure 2: Aggregated Sup35 contributes translation termination activity to [PSI+] cells.
From: Amyloid-associated activity contributes to the severity and toxicity of a prion phenotype
(a) The levels of soluble Sup35 relative to a WT [psi−] strain and stop-codon readthrough relative to WT [PSI+] strain were determined in lysates from WT [psi−] (grey square), WT [PSI+] (white triangle) and [psi−] strains expressing Sup35 from a doxycycline-repressible promoter (Ptet; 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 IB. Error bars represent s.d.; n=6. (b) The relative levels of soluble (black circles) and aggregated (white squares) Sup35 in a Ptet-SUP35 [PSI+] strain were determined at the indicated generations following doxycycline-induced repression of Sup35 synthesis by quantitative IB. Error bars represent s.e.m.; n=3. (c) The relative level of stop-codon readthrough of GST(UGA)DsRedNLS in a Ptet-SUP35 [PSI+] strain was determined by analysis of lysates by quantitative IB for GST at various times after addition of doxycycline. Error bars represent s.e.m.; n=3. (d) [PSI+] (grey) and [psi−] (white) zygotes with repressed Sup35 synthesis were analysed 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 s.e.m.; n≥ 20; *P=0.0001 by unpaired Student’s t-test.
