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. Author manuscript; available in PMC: 2013 Feb 13.
Published in final edited form as: Methods Mol Biol. 2012;821:317–327. doi: 10.1007/978-1-61779-430-8_20

Fig. 2.

Fig. 2

Spleen and lymph node cells were harvested from wild-type and T-Mtor−/− mice. After RBC lysis and extensive washing, splenocytes were stimulated with 3 μ g/mL anti-CD3 and 2 μ g/mL anti-CD28 overnight. Cells were surface stained with anti-CD4–biotin, fixed in 4% formalin, and permeablized with 100% methanol. After a 10-min block in 10% FCS, cells were stained intracellularly for phosphorylation of S6K1 and Akt. Plots are gated on CD4 cells. Voltage was adjusted based on secondary-only controls and gates were set with an unstimulated control. Wild-type cells activate both mTORC1 (pS6K1) and mTORC2 (pAkt) signaling when stimulated, but mTOR-defi cient T cells lack activation of both mTORCs.