Ficoll hypaque

Primary murine T cells (6×10⁷) were labeled on ice for 25 min with 20 μg/ml biotin-conjugated anti-CD3ε (500A2), anti-CD4 (RM4-5) and anti-CD8a antibodies (53-6.7), washed with PBS with 2% FBS, incubated on ice for 25 minutes with 10 μg/ml FITC-streptavidin in the presence or absence of pervanadate as indicated, placed in a 37°C water bath for 2 minutes or for the indicated times, and immediately lysed in cold lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100) containing 100 μM PMSF, Complete™ protease inhibitors (Roche), 1 mM Na₃VO₄ and 25 mM N-ethylmaleimide (Sigma). A stock solution of 50 mM pervanadate was freshly prepared by mixing equal volumes of 100 mM H₂O₂ with 100 mM Na₂VO₄. For proteasomal inhibition, cells were pre-treated with 100 μM MG-132 (Boston Biochem, Inc.) for 1 hr at 37°C, then stimulated as described with MG-132 present during all the steps of the stimulation. Human peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of healthy volunteers by Ficoll-Hypaque (Lymphocyte Separation Medium 1.077, Lonza) gradient centrifugation. Peripheral blood T cells were isolated from PBMC by negative selection using Pan T cell Isolation Kit (Miltenyi Biotec). Primary human T cells (3.5×10⁷) were stimulated as described above using biotin-conjugated CD3ε (UCHT1) antibody and FITC-streptavidin.