Class 1 and Class 3 PI3Ks mediate IGF1-induced SGK3 activity.
HEK293 cells were serum-starved for 16 h. One hour prior to stimulation, cells were left untreated (A and B) or treated with 1 µM VPS34-IN1 (C and D) or 0.5 µM GDC-0941 (E and F) or 1 µM VPS34-IN1 and 0.5 µM GDC-0941 (G and H). Cells were then stimulated with 50 ng/ml IGF1 for the indicated times in the presence of inhibitors. Cells were lysed and endogenous SGK3 was immunoprecipitated with anti-SGK3 antibody. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies, after being assayed for SGK3 kinase activity by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32PγATP in a 30 min reaction. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.