GFP-Vps34 (A) or GFP-UV-RAG (B) knock-in HEK293 cells that stably express either the anti-GFP nanobody (Control-aGFP16) or the VHL-E3-ligase–anti-GFP nanobody (VHL-aGFP16) were generated as described in the Materials and Methods. Cells were serum-starved overnight and treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with 50 ng/ml IGF1 for 15 min. Endogenous SGK3 was immunoprecipitated from cell lysates and kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.
