TCA precipitation and western blotting

Total proteins were prepared with trichloroacetic acid (TCA) precipitation. Cells were grown in 50 ml YP medium containing 2% glucose, and harvested at OD₆₀₀ of 0.8. The cells were suspended in 20% TCA before undergoing cell lysis with glass bead beating. Pellets were collected and washed twice with 5% TCA. The pellets with 5% TCA solution were incubated on ice for 30 min. After removing supernatant, pellets were suspended in SDS sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 4% β-mercaptoethanol, 20% glycerol) containing bromophenol blue (1 mg ml⁻¹). The protein samples were then subjected to western blotting. The protein samples were resolved by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membrane at constant voltage 10 V for 1 h. After rinsing the membranes in Ponceau S solution to check transfer quality, the membranes went through blocking reaction with tris-buffered saline and Tween-20 (TBS-T) buffer (10 mM Tris-HCl, pH 7.5, 3 mM KCl, 150 mM NaCl, 0.1% Tween-20) containing 5% of skim milk for 1 h. The membranes were then incubated in TBS-T buffer containing 5% skim milk with the relevant primary antibodies (see Supplementary Table ²) for 1 h at room temperature. The membranes were washed with TBS-T buffer three times, 5 min each, and were incubated with rabbit secondary antibody (Jackson Lab; 111-035-003). After 45 min of incubation with the secondary antibody in TBS-T buffer containing 5% skim milk, the membranes were washed with TBS-T for four times, 5 min each. The blotted membranes were applied with Immobilon western chemiluminescent HRP substrate (Millipore) to detect blot band in X-ray film. Uncropped scans of the western blot analyses are shown in Supplementary Fig. ¹⁰.