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Plates were washed twice with ice-cold PBS, scraped, pelleted 250 g at 4 C for 3 min and treated on ice for 5 min with 500 l of cytoplasmic lysis buffer (10 mM Tris-Cl, pH 8, 0.32 M Sucrose, 3 mM CaCl 2 , 2 mM MgCl 2 , 0.1 mM EDTA, 0.5 % TritonX-100)

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Although CTLA-4 and PD-1 are part of separate pathways, they exert similar inhibitory effects on effector T cell metabolism, including downregulation of AKT phosphorylation, reduced amino acid uptake, and general suppression of metabolic activity