Protease cocktails

Purified CD4 T cells were lysed with 0.5% NP-40 containing TMSD lysis buffer (40 mM Tris-HCl, pH 7.9, 5 mM MgCl₂, 250 mM sucrose, 1 mM DTT, 0.5% NP-40 [IGEPAL], protease cocktails [Sigma-Aldrich], and phosphatase cocktails [Millipore]) on ice for 15 min. The subcellular fractions were prepared by first centrifugation at 800g for 10 min, to remove nuclei. The supernatant was spun down a second time to remove residual cell and nuclear fragments. This supernatant was used as cytosolic fraction. The nuclei were washed twice with TMSD lysis buffer and then incubated with high-salt lysis buffer (50 mM Tris-HCl, pH 7.9, 420 mM NaCl, 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl₂, 10% glycerol, protease cocktails [Sigma-Aldrich], and phosphatase cocktails [Millipore]) for 10 min on ice. The nuclear extracts were sonicated for 10 cycles (30 s per cycle; Bioruptor Diagenode), followed by centrifugation at 13,000 rpm for 15 min, to obtain the final nuclear extract. Protein concentrations were determined by standard BCA assay. The extracts or whole cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked, incubated with primary antibodies followed by horseradish peroxidase–conjugated secondary antibodies, and developed with ECL systems according to the manufacturer’s instructions.

Fractionated lysates or lysates obtained by using ODG lysis buffer (2% N-octyl-β-d-glucopyranoside, 50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl₂, 10% glycerol, protease cocktails [Sigma-Aldrich], and phosphatase cocktails [Millipore]) were incubated with antibodies cross-linked to protein G dynabeads. The cross-linking of antibodies to beads was achieved by using BS3 cross-linker (Pierce). Immunoprecipitates were washed with lysis buffer and eluted by boiling in sample loading buffer. Coimmunoprecipitates were detected by Western blot analysis.

Purified CD4 T cells were incubated with 10 µg/ml of anti-CD3 antibody and 2.5 µg/ml of anti-CD28 antibody on ice for 20 min, followed by cross-linking with 20 µg/ml of anti–Armenian hamster IgG secondary antibody for 2 min or 10 min at 37°C. For PMA stimulation, cells were incubated with prewarmed media containing 5 ng/ml of PMA for 10 min at 37°C. For stimulation of thymocytes, thymocytes were incubated with 10 µg/ml of anti-CD3 biotinylated antibody and 10 µg/ml of anti-CD4 biotinylated antibody for 20 min on ice, followed by cross-linking with 20 µg/ml of streptavidin. Cells were washed with cold HBSS and then lysed with 0.5% of NP-40 lysis buffer (50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl₂, 10% glycerol, 0.5% NP-40, and protease cocktails [Sigma-Aldrich]) containing phosphatase inhibitory cocktail (Millipore) for 15 min on ice. Lysates were obtained by centrifugation at 13,000 rpm for 15 min to remove insolubles. Phosphorylated Erk1/2 and total Erk were detected by Western blot analysis using rabbit anti–phospho-Erk1/2 (T202/Y204) antibody (D13.14.4E; Cell Signaling Technologies) and mouse anti-Erk antibody (L34F12; Cell Signaling Technologies), respectively.