| TORC1 was purified from RL175-2d or RL176-1b cells treated with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD (250 ml per assay point) to an OD600 of not, vert, similar1.0 were chilled on ice, collected by centrifugation, washed with H2O, resuspended in lysis buffer (1× PBS, 10% [w/v] glycerol, 0.5% [v/v] Tween 20, PI, and PPi), transferred to 2 ml screw-cap tubes half-filled with glass beads (0.5 mm), and disrupted in a Fast Prep machine at 4°C (Bio101; 5× 30 s at max. speed). Crude lysates were cleared of debris with two 1000 × g spins and protein concentrations normalized as necessary. Extracts were precleared over CL-4B Sepharose before 7 μl of IgG Sepharose (GE Healthcare) per assay point was added and the mix rotated for 90 min at 4°C. Beads were collected in a column, washed with cold lysis buffer, and aliquotted to 1.5 ml tubes. Kinase reactions were performed in kinase buffer (1× PBS, 20% glycerol, 0.5% Tween 20, 4 mM MgCl2, 10 mM DTT, 2 μg/ml heparin, and PI [−EDTA]) in a final volume of 30 μl containing not, vert, similar350 ng of recombinant Sch9. Assays were started with the addition of 100 μM ATP and 50 μCi [γ-32P]ATP, shaken for 20 min at 30°C, and terminated with the addition of 8 μl of 5× SDS-PAGE sample buffer. Samples were heated to 95°C for 5 min before being separated by SDS-PAGE, stained with Coomassie, and analyzed using a BioRad Molecular Imager.
| | PPi: 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF. |
| | Cultures were mixed with TCA (final concentration 6%) and put on ice for at least 5 min before cells were pelleted, washed twice with cold acetone, and dried in a speed-vac. Cell lysis was done in 100 μl of urea buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi) with glass beads in a bead beater with subsequent heating for 10 min to 65°C. For NTCB cleavage 30 μl of 0.5 M CHES (pH 10.5) and 20 μl of NTCB (7.5 mM in H2O) were added and samples incubated over night at RT before 1 vol of 2× sample buffer (+20 mM TCEP and 0.5× PPi) was added. Further analysis was done by SDS-PAGE and immunoblotting using anti-HA antibody 12CA5 or anti-T570-P antiserum. |