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. Author manuscript; available in PMC: 2015 Jan 15.
Published in final edited form as: Nat Commun. 2014 Jul 15;5:4384. doi: 10.1038/ncomms5384

Fig. 1. Aggregated Sup35 associates with Sup45 and the ribosome in [PSI+] cells.

Fig. 1

a. PrD-HA expressed in haploid [PSI+] (+) or [psi] (-) yeast strains was immunocaptured (IC) from lysates (Ab, +) in comparison with a reaction lacking specific antiserum (Ab, -), and the isolated complexes were analyzed by SDS-PAGE and immunoblotting for PrD, Sup35 and Sup45. Total protein in the cell lysates was analyzed in parallel to ensure equivalent expression and extraction. b. Following PrD-HA IC as in panel (a), the presence of ribosomal subunits was assessed by qRT-PCR using primers specific for either 18S or 26S ribosomal RNAs. All strains express untagged full-length Sup35 except lane 6, which contains a Sup35 deletion. Lanes 6 and 7 also express the GTPase domain of Sup35 (Sup35-C). The data represent the average of at least three independent experiments, and the error bars represent SEM; *p=0.013; **p≤0.004 by unpaired Student’s t-test. c. Sucrose density gradients were used to fractionate lysates isolated from wild-type (WT) [psi] or [PSI+] strains and from [PSI+] strains expressing the PrD in the presence of Sup35-C or full-length Sup35. The lysates were either untreated (-) or treated with RNase (+) before fractionation. Following centrifugation, fourteen fractions including the pellet were collected and analyzed by immunoblotting (IB) for Sup35 or the PrD.