Protease inhibitor cocktail set 4

Desired strains were grown to OD₆₀₀ = 1 for asynchronous cultures or to OD₆₀₀ = 0.5, supplemented with 10 µg/ml final concentration α-factor mating pheromone, 0.2 M (final concentration) hydroxyurea (Sigma-Aldrich), or 15 µg/ml (final concentration) nocodazole (Sigma-Aldrich) and grown for another 2 h 15 min to OD₆₀₀ = 1. Rapamycin (LC Laboratories) and cycloheximide (Sigma-Aldrich) at a final concentration of 200 nM and 25 µg/ml, respectively was added to a culture of OD₆₀₀ = 1 30 min before harvesting. In case of nutrient withdrawal, cells were grown to OD₆₀₀ = 1, spun down, and resuspended in H₂O and then grown another 45 min before harvest. Cells were harvested and either frozen as droplets in liquid N₂ and subsequently lysed with a freezer mill or lysed using glass beads in a mini BeadBeater (Biospec Products). The yeast pellets were dissolved in buffer A (25 mM Hepes, pH 8.0, 2 mM MgCl₂, 0.5 mM EGTA, pH 8.0, 0.1 mM EDTA, 0.1% NP-40 [EMD Millipore], 15% glycerol, 150 mM KCl, and 1× protease inhibitor cocktail set IV [EMD Millipore]). The cleared lysate was incubated for 2 h with magnetic DynaBeads (Thermo Fisher Scientific) coupled with α-FLAG M2 antibody (Sigma-Aldrich). Beads were washed three times with buffer B (25 mM Hepes, pH 8.0, and 150 mM KCl) before the beads were resuspended in sample buffer.