Author: Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.; Carter-Su, Christin
Title: Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics Document date: 2012_5_8
ID: xtj2ywf3_13
Snippet: 3T3-F442A preadipocytes (15-cm culture dishes) were incubated in serum-free medium overnight before GH treatment. After GH treatment, cells were washed with PBSV, solubilized with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate supplemented with 1 mM Na 3 VO 4 , 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupeptin for 10 min on ice, and then centrifuged for 10 min at 16,000 Ï« g at .....
Document: 3T3-F442A preadipocytes (15-cm culture dishes) were incubated in serum-free medium overnight before GH treatment. After GH treatment, cells were washed with PBSV, solubilized with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate supplemented with 1 mM Na 3 VO 4 , 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupeptin for 10 min on ice, and then centrifuged for 10 min at 16,000 Ï« g at 4 C. Supernatants were incubated with â£NHE1 and protein A-agarose beads overnight at 4 C. Beads were washed with lysis buffer, an 80:20 mixture of lysis buffer:SDS-PAGE sample buffer was added, and the samples were boiled for 5 min. Proteins were subjected to SDS-PAGE and immunoblotted as above. Band intensity was quantified using Odyssey software (LI-COR, version 2.0).
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