Jurkat T cells were rinsed with RPMI and resuspended at 5 × 106 cells/ml and rested for 15 min at 37°C. Cells were treated with anti-TCR antibody (C305, 0.85 μg/ml) or CskAS inhibitor 3-IB-PP1 (provided by the Shokat laboratory), PP2 (Tocris), or dimethyl sulfoxide (DMSO). Cells were then lysed through addition of lysis buffer containing a final concentration of the following: 1% NP-40, NaVO4 (2 mM), NaF (10 mM), EDTA (5 mM), phenylmethylsulfonyl fluoride (2 mM), aprotinin (10 μg/ml), pepstatin (1 μg/ml), leupeptin (1 μg/ml), and PP2 (25 μM). Lysates were placed on ice, and debris were pelleted at 13,000g. Primary T cells were resuspended at 20 to 40 × 106 cells/ml and lysed using 6× SDS–polyacrylamide gel electrophoresis sample buffer. DNA was pelleted in an ultracentrifuge at 70,000 rpm. Supernatants were run on 4 to 12% NuPage or 10% bis-tris gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated with blocking buffer [2% bovine serum albumin tris-buffered saline with 0.1% Tween-20 (TBS-T)] and then probed with primary antibodies overnight at 4°C. The following day, blots were rinsed and incubated with horseradish peroxidase–conjugated secondary antibodies (diluted 1:5000). All antibodies used for immunoblotting were diluted 1:2000 in blocking buffer unless otherwise stated. Blots were detected using chemiluminescent substrate and a ChemiDoc (Bio-Rad) or iBright (Invitrogen) imaging system. Phosphorylation was assessed at 2 min after acute treatment with inhibitor or anti-TCR stimulation unless otherwise stated. Quantification was performed using Image Lab or iBright analysis software.

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