T5168 monoclonal anti a tubulin clone b 5 1 2

WT and mutant strains were grown to the exponential phase (OD600, 1.0) in 20 ml EMM2. Yeasts were harvested by centrifugation (2,250× g for 5 min), and the pellet was used to analyze protein aggregation as described previously (Rand and Grant, 2006 (link)). Briefly, spheroplasts were pelleted and resuspended in lysis buffer [50 mM potassium phosphate buffer, pH 7, 1 mM EDTA, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride and Complete Mini protease inhibitor cocktail (Roche)]. Cell disruption was carried out by three vortex cycles (1 min of vortex and 1 min on ice) with 220 mg of acid-washed glass beads (Sigma-Aldrich G8772). Membrane proteins were removed by washing twice with 320 μl lysis buffer and 80 μl 10% NP-40 (Sigma-Aldrich), and the final aggregated protein extract was resuspended in 20 μl 1X loading buffer. Total and aggregated protein extracts were analyzed by Western blotting using an anti His-tag antibody (His Tag Antibody MAB050R-100, R&D Systems). Western blots against tubulin were performed as internal controls using T5168 monoclonal anti-a-tubulin clone B-5-1-2 (Sigma-Aldrich). Bands corresponding to WT, mutants, and tubulin (for total protein), or WT and mutants (for aggregated proteins) were quantified using ImageJ software.

Wild-type and mutant strains were grown at the exponential phase (OD600, 1.0) in 20 ml of EMM2 medium at 30°C with constant agitation. Yeasts were harvested by centrifugation at 2,250 × g for 5 min at room temperature, and the pellet was used to analyze protein aggregation as described previously (Rand and Grant, 2006 (link)). Briefly, cells were pelleted and resuspended in lysis buffer [50 mM potassium phosphate buffer, pH 7, 1 mM EDTA, 5% glycerol, 1 mM of phenylmethylsulfonyl fluoride, and Complete Mini protease inhibitor cocktail (Roche)]. Cell disruption was carried out by three vortex cycles (1 min of vortex and 1 min on ice) with 220 mg of acid-washed glass beads (Sigma-Aldrich G8772). Membrane proteins were removed by washing twice with 320 μl lysis buffer and 80 μl of 10% NP-40 (Sigma-Aldrich), and the final aggregated protein extract was resuspended in 20 μl of 1X Laemmli sample buffer. Total and aggregated protein extracts were analyzed by Western blot using an anti His-tag antibody in a 1:1,000 dilution (His Tag Antibody MAB050R-100, R&D Systems). Western blot against tubulin was performed as an internal control (T5168 monoclonal anti-a-tubulin clone B-5-1-2, Sigma-Aldrich). Bands representing WT, the pREP41_tRNA strain, and tubulin (for total protein), or WT and pREP41_tRNA strain (for aggregated proteins) were quantified using ImageJ software.