Total lat

Cellular lysates were mixed with Laemmli sample buffer, heated at 95 °C for 10 minutes and separated on NuPAGE Bis-Tris gels by electrophoresis and transferred to nitrocellulose membranes using the iBlot transfer system (Thermo Scientific). Membranes were incubated in 3% fat-free dry milk for 1 hour at room temperature followed by overnight incubation with primary antibodies. Proteins were detected with Super Signal West Dura (Thermo Scientific #34075) using a Bio-Rad ChemiDoc MP imaging system. Immunoblots were quantified using ImageJ (NIH). Primary antibodies used were: pLck (Y394, R&D Systems), pZAP-70 (Y319), AP1, NFĸB and Lck from Cell Signaling, pLAT(Y226), total ZAP-70, and NFAT-1 from BD Biosciences, total LAT from Biolegend, beta-Actin from Sigma, CD3 (OKT3), and GAPDH from Thermo Fisher, CD28 from BD Biosciences and acetyl histone H3K9 and trimethyl histone H3K27 from Millipore.

IL-2 cytokine released into cell culture supernatant was quantified using human IL-2 ELISA kit (BD Biosciences) according to the manufacturer’s instructions. Jurkat cells were stimulated with anti-CD3 (5 μg/ml) for the indicated times prior to addition of cell lysis buffer (Cell Signaling). Following PMA/Ionomycin stimulation for 15 minutes, nuclear proteins were isolated using nuclear protein isolation kit (NEPER, Thermo Scientific) following manufacturer’s instructions. Proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (BIORAD). Membranes were incubated in protein-free blocking buffer (Thermo Scientific) for 1 hour at room temperature followed by incubation with primary antibodies. Proteins were detected with Amersham ECL (GE Healthcare) using a Kodak Imager. Primary antibodies used were: NFAT and pLAT(Y226; BD Biosciences); total LAT (Biolegend); pZAP70 (Y319); total ZAP70; pLck (Y394/ pSrcY416); total Lck (Y394); Csk; YY1 (all from Cell Signaling Technology); PTPRE (Origene, clone 4B2). Immunoblots were quantified by ImageJ.