Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics

Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.; Carter-Su, Christin

3T3-F442A preadipocytes were grown in DMEM supplemented with 1 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin, and 8% calf serum. Cells were incubated in serum-free medium overnight before GH treatment. In the inhibitor experiments, cells were pretreated with 100 nM wortmannin (Sigma, St. Louis, MO) and/or 100 nM CL-1040 (MEK inhibitor) (Selleck Chemicals, Houston, TX) for 30 min before GH treatment. After GH treatment, cells were washed with PBSV. Cells were solubilized with a buffer containing four parts lysis buffer [50 mM Tris (pH 7.5), 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA, supplemented with 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 g/ml aprotinin, 10 g/ml leupeptin (pH 7.5)] and one part SDS-PAGE sample buffer [50 mM Tris, 1% sodium dodecyl sulfate, 0.001% bromophenol blue, 10% glycerol, and 270 mM ␤-mercaptoethanol (pH 6.8)]. Samples were boiled and subjected to SDS-PAGE. Proteins in the gel were transferred to nitrocellulose, detected by immunoblotting with the indicated antibody, and visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). For experiments assessing raptor and phospho-raptor, cells were solubilized with lysis buffer, and cell lysates were clarified by centrifugation (10 min, 16,000 ϫ g, 4 C) before SDS-PAGE.