Figure 3.

D4476 specifically inhibits the phosphorylation at Ser322 and Ser325 specifically in H4IIE cells. Cells were serum starved for 4 h, and then stimulated for 30 min without (−) or with (+) 20 nM insulin. Aliquots of the cell lysates (2.5 mg protein) were then immunoprecipitated with 20 μg of anti-FOXO1a antibody raised against the whole protein. After washing the immunoprecipitates and denaturation in SDS, aliquots of the solubilized material were subjected to SDS–polyacrylamide gel electrophoresis. Following transfer to nitrocellulose, the membranes were subjected to immunoblotting using the antibodies used in Fig 1, as well as with a phospho-specific antibody that recognizes Ser329 (pSer329). Where indicated, the cells were incubated for 60 min with various concentrations of D4476 prior to stimulation with insulin. Similar results were obtained in at least six independent experiments.