Changes

TORC1 was purified from RL175PPi: 10 mM NaF, 10 mM NaN3, 10 mM p-2d or RL176nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-1b cells treated glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF. Cultures were mixed with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD TCA (250 ml per assay pointfinal concentration 6%) to an OD600 of not, vert, similar1.0 were chilled and put on ice, collected by centrifugationfor at least 5 min before cells were pelleted, washed twice with H2Ocold acetone, resuspended and dried in a speed-vac. Cell lysis was done in 100 μl of urea buffer (1× PBS, 10% 50 mM Tris [w/vpH 7.5] glycerol, 0.5mM EDTA, 6 M urea, 1% [v/v] Tween 20SDS, PI1 mM PMSF, and 0.5× PPi), transferred to 2 ml screw-cap tubes half-filled with glass beads (in a bead beater with subsequent heating for 10 min to 65°C. For NTCB cleavage 30 μl of 0.5 mm), and disrupted in a Fast Prep machine at 4°C M CHES (Bio101; 5× 30 s at maxpH 10. speed5). 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 20 μl of IgG Sepharose NTCB (GE Healthcare7.5 mM in H2O) per assay point was were added and the mix rotated for 90 min samples incubated over night at 4°C. Beads were collected in a column, washed with cold lysis buffer, and aliquotted to RT before 1.5 ml tubes. Kinase reactions were performed in kinase vol of 2× sample buffer (1× PBS, +20% glycerol, 0.5% Tween 20, 4 mM MgCl2, 10 mM DTT, 2 μg/ml heparin, TCEP and PI [−EDTA]) in a final volume of 30 μl containing not, vert, similar350 ng of recombinant Sch90. 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 bufferPPi) was added. Samples were heated to 95°C for 5 min before being separated Further analysis was done by SDS-PAGE, stained with Coomassie, and analyzed immunoblotting using a BioRad Molecular Imageranti-HA antibody 12CA5 or anti-T570-P antiserum.