Aliquots of transduced Prkcq−/− CD4+ T cells (5 × 104) were stimulated for 48 hours with CD3 or CD28 mAbs, or both, and IL-2 concentration in culture supernatants was determined by ELISA, according to the manufacturer’s instructions (BioLegend). A 96-well plate (Corning Costar) was coated overnight at 4°C with an IL-2 mAb. Triplicates of IL-2 standards and supernatants from cultured cells were then added to the plate, followed by a 2-hour incubation at room temperature. A biotinylated IL-2 Ab was added to the plate, followed by incubation for 1 hour, and then, streptavidin-HRP was added, followed by incubation for 30 min, all at room temperature. After washing, the amount of bound avidin was assessed with TMB peroxidase that was acidified by 2 N H2SO4. Absorbance at 450 nm was measured in a SpectraMax plate reader (Molecular Devices).

For peptide ELISA, the following N-terminal biotinylated peptides were synthesized and purified to >90% as judged by high-performance liquid chromatography (HPLC) and MS (GenScript): Y126, AMVRDYVRQTWK; pY126, AMVRDpYVRQTWK; Y292, LNSDGYTPEPAR; pY292, LNSDGpYTPEPAR; Y315/319, TSVYESPYSDPE; pY315, TSVpYESPYSDPE; pY319, TSVYESPpYSDPE; and 2pY (pY315 and pY319), TSVpYESPpYSDPE. Biotinylated peptides (10 μg/ml in 100 μl of PBS) were added to microtiter wells of a streptavidin-precoated plate (Pierce) and incubated overnight at 4°C. Proper and equal binding of the different peptides to the wells was confirmed by adding HRP-streptavidin to a duplicate set of wells. Nonspecific binding sites were blocked by incubation with 300 μl of 5% BSA/PBS for 1 hour at room temperature. After washing with PBST (0.5% Tween 20/PBS), GST or GST-C2 proteins [1 μg in 1% (w/v) NP-40 lysis buffer] were added for 1 hour. After further washing, the wells were incubated for an additional hour with a mouse Ab against GST (1:2000 in PBST), followed by a 1-hour incubation with HRP-conjugated anti-mouse Ig at room temperature. Samples were analyzed in triplicate in both peptide-coated and uncoated control wells, and ELISA readout was performed as described above.

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