Fig. 3. The severity and toxicity of the [PSI⁺] state cannot be explained by fluctuations in the level of soluble Sup35.

a. The levels of soluble Sup35 were determined in lysates from wildtype [psi] and [PSI⁺] strains and from [PSI⁺] strains expressing full-length Sup35 from P_tet at ~1.5-fold the wildtype level (Sup35) or the PrD from P_SUP35 (PrD) by quantitative immunoblotting for Sup35. All strains also expressed the stop-codon readthrough reporter GST(UGA)DsRedNLS. Error bars represent SEM; n≥3. b. Strains expressing full-length Sup35 from P_SUP35 and Sup35-C from P_tet were transformed with either vector or a high-copy plasmid that over-expresses the PrD ~10-fold on medium lacking uracil (-Ura) or ~100-fold on medium lacking leucine (-Leu). The levels of soluble Sup35 under these conditions were determined in lysates by quantitative immunoblotting. Error bars represent SEM; n≥5; *p=0.004 by unpaired Student’s t-test. c. Stop-codon readthrough was determined in lysates from the strains described in panel (a) by quantitative immunoblotting for GST. Error bars represent SEM; n≥3; *p=0.017 by unpaired Student’s t-test.. d. Stop-codon readthrough was determined as in panel (c) in lysates from the strains described in panel (b). Strains were grown in medium lacking uracil (-Ura) in the presence of doxycycline to repress expression of Sup35-C. Error bars represent SEM; n≥3; *p=0.01 by unpaired Student’s t-test.. The differences in levels of stop codon readthrough in panels (c) and (d) represent the different baselines in rich (c) and minimal (d) medium. e. The strains described in panel (b) were plated on medium lacking uracil (-Ura) or lacking leucine (-Leu) in the absence (-) or presence (+) of doxycycline to repress Sup35-C expression and assess toxicity of overexpressed Sup35.