T Cell Stimulation.
For T cell activation, 2 × 107 Jurkat E6, JCam-1, or JCam-1-Lck cells (20) cultured in RPM1 1640 plus 10% fetal bovine serum and 2 mM l-glutamine penicillin/streptomycin (GIBCO BRL) were serum starved overnight and then stimulated for varying times with 10 μg/ml anti-CD3 and 5 μg/ml anti-CD28 antibodies. Alternatively, 2 × 107 murine thymocytes or lymph node T cells were incubated with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 antibodies on ice for 30 min followed by cross-linking with 5 μg anti–hamster IgG for varying times at 37°C. Aliquots of cell cultures were subjected to anti-pTyr immunoblotting analysis to confirm cell activation.