Endosome isolation and in vitro mTORC1 kinase activity assay

Endosomes were isolated from naïve CD4⁺ T cells on day 3 after activation when mTORC1 activity peaked, by using an endosome isolation kit (ED-028, Invent Biotechnologies) according to manufacturer’s instructions. In some experiments, cells were pre-treated for 2 hours with 1 μM AKT inhibitor (MK-2206 2HCl, Selleckchem) prior to harvesting. Briefly, 2 × 10⁷ cells were suspended in lysis buffer A (supplemented with protease inhibitor cocktail) and cell extracts were filtered to remove intact cells, larger organelles and plasma membranes. The flow-through cell lysates were mixed with the supplied precipitation buffer B followed by centrifugation. After centrifugation, endosomes were enriched in pellets. Supernatants were discarded, and the endosome pellets were resuspended in RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology). Protein concentrations were measured using BCA Protein Assay Kit (23227, Pierce) and equal amounts of protein were loaded for immunoblotting analysis of indicated markers. Alternatively, the endosome pellets were resuspended in mTORC1 kinase buffer (25 mM HEPES, 50 mM KCl, 10 mM MgCl₂, 20% glycerol, 4 mM MnCl₂ and 250 μM ATP) prepared according to a previous study (56) and divided into aliquots of 10 μl each containing 10 μg endosomes. 100 ng of S6K1 Human Recombinant Protein (TP317324, Origene) diluted in 5 μl of mTORC1 kinase buffer was added as the substrate, and the Kinase assays were performed at 30°C for 20 min in a final volume of 15 μl. Reactions were stopped by adding 5 μl of 4 x sample buffer and loaded for immunoblotting analysis.