Yeast protease inhibitor cocktail

Cells expressing an HA-tagged version of Csr2 (endogenously tagged at its genomic locus; ySL1037) were grown overnight at 30°C in glucose-containing SC medium to an A₆₀₀ of 0.3–0.6 and transferred to SC-lactate medium for 4 h at 30°C. Cells from half of the culture (∼400 OD units) were collected by centrifugation (5 min, 4,000 g, 4°C). Glucose (2% final) was added to the other half, and cells were incubated for 5 min at 30°C before being harvested. Each pellet was resuspended in 4 ml lysis buffer (200 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM DTT, and 0.1% Triton X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [Sigma-Aldrich], 10 mM N-ethylmaleimide [Sigma-Aldrich], 100 µM MG-132 [Enzo Life Sciences], and 1% yeast protease inhibitor cocktail [P8215; Sigma-Aldrich]). Cells were mechanically disrupted with glass beads on a vortex at 4°C for 4 × 30 s. The lysate was then cleared at 3,000 g for 5 min, at 4°C, and the supernatant was incubated with 25 µl anti-HA affinity resin (Roche) for 1 h 30 min at 4°C under rotation. The beads were then washed three times with lysis buffer and once with H₂O and resuspended in 100 µl H₂O, before being subjected to trypsin digestion and mass spectrometry analysis.

Yeast lysates were prepared by the alkaline lysis procedure and then neutralized as described for the yeast WB. The lysates were incubated with anti-HA antibody in yeast IP lysis buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1% NP-40, 0.2% Triton X-100, 5% glycerol, 1 mM DTT, 20 mM N-ethylmaleimide, and yeast protease inhibitor cocktail (Sigma Aldrich, P8215) at 4°C overnight. Protein A/G PLUS-agarose slurry (50 μl; Santa Cruz Biotechnology) was added and incubated with the lysates for another 4 hours. After centrifugation, immunoprecipitates were washed with yeast IP lysis buffer two times and then resuspended in 2× Laemmli buffer for SDS-PAGE and immunoblotting with indicated antibodies.