Figure 4.

Sup35PrD-GFP and endogenous Sup35 SDS-resistant oligomers show change in migration over time. (a) Sup35PrD-GFP was over expressed for 8, 16 and 24 hours. Cultures were lysed and immediately subjected to SDD-AGE-immunoblot. [psi ^-], established [PSI ⁺], and uninduced strains carrying the Sup35PrD-GFP plasmid were run as controls. Samples were run on the same agarose gel and separated for immunoblotting. The blots were incubated with either anti-GFP (left) or anti-Sup35C antibody (right). Anti-GFP antibody exclusively labels the overproduced Sup35PrD-GFP protein whereas Sup35C antibody exclusively labels the endogenous Sup35p protein. Blots were aligned in the figure based on the migration of bands in the marker lane. The migration of the monomeric endogenous Sup35p band from [psi ⁻] cultures and the oligomeric smear from [PSI ⁺] cultures is indicated on the right. While all cultures were lysed and immediately loaded onto SDD-AGE, the [PSI ⁺] control was stored for four days at −80 °C, which likely explains the minor degradation products. Replicate blots showed that fresh [PSI ⁺] lysates lacked any degradation products. (b) Similarly induced cultures were boiled or unboiled and run on a 7% SDS PAGE, and immunoblotted with anti-GFP antibody. The molecular weights (kDa) of the marker is as indicated on the left. Monomeric Sup35PrD-GFP migrates at approximately 55 kDa. (c) The top panel is a shorter exposure of the same blot from B (anti-GFP), showing the monomeric Sup35PrD-GFP bands. The same blot was re-immunolabeled with anti-Sup35 (middle) and anti-PGK (bottom) as indicated.