L., Yang J. the indicated antibodies were added to the cleared cellular lysates and incubated with rotation for 6C16 h. Then 25 l of protein G-agarose were added, and the incubation was continued for 1 h. Immunoprecipitates captured with protein G-agarose were washed 3 times with the CHAPS lysis buffer and twice by wash buffer A (50 mmol/liter HEPES, (pH 7.5), 150 mmol/liter NaCl) and boiled in 4 SDS sample buffer for Western blot. In Vitro mTOR Kinase Assay Transfected HEK293T cells were produced in 100-mm dishes for 48 h in DMEM made up of 10% FBS and lysed in 1 ml of lysis buffer with 0.3% CHAPS. Half of total cell lysate was incubated with anti-mTOR or FLAG antibody for 3 h followed by another hour of incubation with 25 l of protein G-agarose beads. Immunoprecipitates were washed twice by lysis buffer, twice by wash buffer B (20 mmol/liter Tris (pH 7.5), 500 mmol/liter NaCl, 1 mmol/liter EDTA, 20 mmol/liter -glycerophosphate, 5 mmol/liter EGTA, 1 mmol/liter DTT, 1 mmol/liter orthovanadate, 40 mg/ml phenylmethylsulfonyl fluoride (PMSF), 10 g/ml leupeptin, 5 g/ml pepstatin), once with wash buffer C (10 mmol/liter HEPES (pH 7.4), 50 mmol/liter glycerophosphate, 50 mmol/liter NaCl, 1 mmol/liter DTT, 1 mmol/liter orthovanadate, 40 mg/ml PMSF, 10 g/ml leupeptin, 5 g/ml pepstatin), and once with mTOR kinase assay buffer without ATP (10 mmol/liter HEPES (pH 7.4), 50 mmol/liter NaCl, 50 mmol/liter glycerophosphate, 1 mmol/liter DTT, 10 mmol/liter MgCl2, 4 mmol/liter MnCl2). Kinase assay toward recombinant GST-S6K1 (amino acids 308C400) in washed immunoprecipitates was done for 30 min at 30 C in 30 l of mTOR kinase buffer with 100 mol/liter ATP unlabeled and 10 Ci [-32P]ATP (PerkinElmer Life Sciences). To stop the reaction, 6 l of 4 SDS sample buffer was added PROTAC Sirt2 Degrader-1 to each reaction, which was boiled for 10 min. The reaction was then separated by 4C12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. 32P incorporated into GST-S6K was assessed by autoradiography. In a cold kinase assay to GST-S6K, phosphorylation S6K was detected by phosphor-S6K-Thr-389 antibody. In Vitro IKK Kinase Assay To map the phosphorylation sites in mTOR, the fragments of the mTOR coding sequence were cloned into pGEX vector (GE Healthcare). Purified GST-mTOR fusion proteins were immobilized to glutathione-agarose for kinase assay. Kinase assays were performed PROTAC Sirt2 Degrader-1 following a previously described protocol (Upstate Biotechnology). Kinase activity was determined by incubating purified IKK with GST-mTOR fragments, GST-IB, or immunoprecipitates of mTOR and Raptor from HEK 293T cells as indicated in the presence of 1 Ci ml?1 [-32P]ATP or cold ATP (100 m) for 30 min at 30 C. Reactions were resolved by SDS-PAGE (4C12%) and processed for autoradiography or protein immunoblotting. Mass Spectrometric PROTAC Sirt2 Degrader-1 Analysis to Identify mTOR Phosphorylation Sites Purified GST-mTOR-(1351C1650) protein was incubated with recombinant IKK for cold kinase assay, resolved by SDS-PAGE, and Rabbit Polyclonal to Serpin B5 stained with Coomassie Blue, excised from the gel, digested with trypsin, and analyzed by tandem mass spectrometry by Dr. John Asara of the Beth Israel Deaconess Medical Center. Cell Proliferation Assays The indicated transfected cells were plated in 6-well plates at a density of 2.0 104 cells/well. Cells were trypsinized and counted using a hematocytometer every day until confluency. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed according to the manufacturer’s recommendations (Promega, Madison, WI). The cells were plated in 96-well microtiter plates at a density of 1 1.0 103 cells/well.