Immunoprecipitation and western blotting

SKG or BALB/c T cells (3.0 £ 10) were stimulated for 72 h with plate-bound anti-CD3 mAb (2C11) in the presence or absence of plate-bound anti-CD28 mAb in RPMI-1640 medium supplemented with 10% fetal calf serum and 50 mM 2-mercaptoethanol. Cells were also stimulated with TPA (1.4 ng ml) and ionomycin (0.14 mM). KJ1.26þ T cells from DO or DO.SKG mice were stimulated with ovalbumin (323–339) peptide in the presence of X-irradiated syngeneic spleen cells (10 cells) as antigen-presenting cells in 96-well round-bottomed plates. The incorporation of [H]thymidine by proliferating lymphocytes during the last 6 h of the culture was measured.