Assay of mTORC1/2 in vitro kinase activity

RAPTOR or RICTOR immunoprecipitates from insulin (100 nM) stimulated HEK293 cells were washed in kinase buffer (25 mM HEPES pH 7.4, 20 mM KCl, and 10 mM MgCl2) and 50 ng of recombinant GST-WAVE2 or GST alone were added to each sample. After 30 min, the complexes were incubated for 30 min in mTOR kinase buffer (25 mM HEPES, 50 mM KCl, 10 mM MgCl2, and 500 μM ATP) containing 100 ng inactive 4E-BP1 (for mTORC1) or 100 ng inactive AKT (for mTORC2) substrate. Reactions were stopped after 30 min by adding 30 μl of sample buffer and boiling for 5 min and then resolved by SDS-PAGE followed by immunoblotting with anti-phospho-4E-BP1 or anti-phospho-AKT (Ser⁴⁷³) antibodies. Estimates of kinase activities were based on the ratio of phosphorylated substrate to total inactive substrate in each reaction. Densitometric analysis of protein bands was performed using Image J and the relative gray values of specific bands calculated and normalized to control values. Additional reagent and antibody information including dilutions, and concentrations can be found in table S1.