Western blot

Purified naïve CD8⁺ T cells cultured under the conditions indicated were harvested, washed with ice-cold PBS, and lysed on ice for 15 to 30 min in a lysis buffer [20 mM tris (pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, aprotinin (1 μg/ml), and leupeptin]. Cell lysates were resolved by 4 to 12% bis-tris SDS–polyacrylamide gel electrophoresis gel (Invitrogen), transferred onto nitrocellulose membrane (Invitrogen), blocked with 5% dry nonfat milk in tris-buffered saline (pH 7.4) containing 0.1% Tween 20, and probed with the following Abs to (Abs were used at 1:1000 and purchased from Cell Signaling Technology unless otherwise described): p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴; D13.14.4E), p-PLCγ (Tyr⁷⁸³; rabbit polyclonal), p-ZAP70 (Tyr³¹⁹; 65E4), p-AKT (Thr³⁰⁸; D25E6), p-AKT (Ser⁴⁷³; D9E), p-p38 (Thr¹⁸⁰/Tyr¹⁸²; D3F9), p-S6K (Thr³⁸⁹; rabbit polyclonal), p-S6 (Ser^235/236; D57.2.2E), p-4EBP-1 (Thr^37/46; 236B4), c-Myc (E5Q6W), RagD (polyclonal; Novus Biotechnology), p-STAT2 (Tyr⁶⁹⁰; D3P2P), p-STAT3 (Tyr⁷⁰⁵; D3A7), p-STAT4 (Try⁶⁹³; D2E4), p-STAT5A/B (Tyr^694/699; A11W; Millipore), and β-actin (AC-15, AC-74; Sigma-Aldrich). Immunoreactivity was detected by enhanced chemiluminescence detection system according to the manufacturer’s instructions (GE Healthcare).