Immunoprecipitation and Immunoblotting.
To prepare cell lysates, 2 × 107 Jurkat cells, thymocytes, or lymph node T cells were suspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM PMSF, 1 mM Na3VO4) and 1 μg/ml each aprotinin, leupeptin, and pepstatin (Amersham Biosciences). After 30 min of incubation on ice, unlysed cells were removed by centrifugation and 100–250 μg of the lysate proteins were then incubated with Protein A sepharose 6B beads (Amersham Biosciences) for 30 min at 4°C followed by 2 h of incubation with specific antibody or rabbit preimmune serum. The immune complexes were then collected over Protein A sepharose and eluted from the beads by boiling in Laemmli buffer. For immunoblotting analyses, immunoprecipitated proteins were suspended in loading buffer, electrophoresed through 10% polyacrylamide gels, and transferred to nitrocellulose membrane. After blocking with 5% skim milk powder in 1× TBST (20 mM Tris-HCl, pH 7.5, 0.9% NaCl, 0.1% Tween 20), membranes were incubated for 1 h with the primary antibody followed by a 1-h incubation with a 1/3,000 diluted horseradish peroxidase–conjugated secondary antibody (Bio-Rad Laboratories) and for 1 min with ECL substrate (Amersham Biosciences) followed by 1–10 min of exposure to Eastman Kodak Co. X-ray film.