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

To validate the results of our phosphoproteomic analysis, cell lysates from 3T3-F442A preadipocytes were treated with GH for varying amounts of time and then blotted with the appropriate phosphospecific antibodies. Immunoblotting with phosphospecific antibodies confirmed a rapid and robust GH-dependent phosphorylation of Shc Tyr423 (Fig. 5A) , Ser184 and Thr247 in PRAS40 (Fig. 5B) , and raptor Ser863 (Fig. 5C) . A rapid, modest GH-dependent phosphorylation of Ser455 in ACLY (Fig. 6A) and Ser 330 in NDRG1 (Fig. 6B) was also observed. Similarly, when NHE1 was immunoprecipitated from 3T3-F442A preadipocytes that had been treated with GH for 0 or 15 min and immunoblotted with ␣pSer707-NHE1, a GH-dependent increase in phosphorylation of Ser707 was observed (Fig. 6C) . In each case, blotting with antibody against the corresponding total protein confirmed that the change in signal was due to a change in the amount of protein phosphorylated and not in the amount of protein.