Fig 3. [SWI⁺] prion is a key determinant of nonsense suppression in [NSI⁺] strains.

(A) Sedimentation analysis of Swi1(1–297)-YFP protein from the 4-1-1-D931 [NSI⁺] and 1-4-1-1-D931 [nsi^-] strains expressing pCUP1-SWI1(1–297)-YFP (URA3) plasmid. Soluble (S) and insoluble (I) fractions were obtained as indicated in Materials and Methods. Swi1(1–297)-YFP was detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). Next, SDD-AGE analysis of insoluble fractions of [NSI⁺] and [nsi^-] strains comprising Swi1(1–297)-YFP was performed. (B) The effects of SWI1 deletion on the [NSI⁺] phenotypic manifestation. SWI1 deletion was obtained as described in Materials and Methods. To express SWI1, the 11-1-1-D931 [NSI⁺] swi1Δ strain was transformed with the YGPM19p21 plasmid from the YSC4613 genomic library, containing a genomic fragment encoding SWI1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with an empty vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO₄ and replica-plated on–Leu–Ade or–Leu Gal media with 150 μM CuSO₄. Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.