Protein expression and western analysis

Cell lysates were prepared as described (Reidy et al. 2014). Briefly, harvested cells were suspended in tris-HCl lysis buffer and broken by agitation with zirconium beads using a BioSpec bead beater. Protein concentration was measured using Bradford reagent. For western analysis, 10–15 µg of protein from cell lysates was separated on 4–20% SDS-PAGE gels and transferred to PVDF membranes. Primary antibodies were rabbit anti-Sis1 raised against an epitope in the carboxy-terminal domain of Sis1 and rabbit anti-Sup35 (a gift from Sue Liebman). Blotted membranes were subsequently stained with amido-black (Sigma # A-8181) as a loading and transfer control.