Loss of mTORC2 activity ablates IGF1-induced SGK3 activity.
Wild-type and SIN1 knock-out HEK293 cells were serum-starved overnight prior to 50 ng/ml IGF1 stimulation for 15 min. Endogenous SGK3 was immunoprecipitated from the lysates and SGK3 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.