Western Blot Analysis of Yeast Proteins

The assay was performed as previously described (Hildebrandt, Cheng et al. 2016 (link); Berger, Kim et al. 2018 (link); Schey, Buttery et al. 2021 (link)). Whole cell lysates of mid log cells were prepared and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 6% stacking with 9.5% resolving gel) then transferred onto nitrocellulose. Blots were blocked with 5% milk then sequentially incubated with rabbit anti-Ydj1 primary antibody (courtesy of A. Caplan) and HRP-conjugated goat antirabbit secondary antibody (Kindle Biosciences, Greenwich, CT). Fluorescence was detected using the KwikQuant Western Blot Detection Kit (Kindle Biosciences) and a KwikQuant Imager and as per manufacturer's instructions. Protein bands were quantified using NIH ImageJ, and resulting values were used for calculating ratios for prenylated and nonprenylated bands. The blots containing yeast Nap1 were treated similarly but incubated with mouse anti-His primary antibody (Thermo Fisher Scientific, Waltham, MA) and HRP-conjugated sheep antimouse secondary antibody (Kindle Biosciences, Greenwich, CT).