Activation of MAPKs in human primary CD4+ T-cells by costimulatory receptors. Human CD4+ T cells were seeded at a concentration of 1 × 106/ml and stimulated with Dynal beads coated with α-CD3/B7-2 Fc (A) or α-CD3/B7-H2 Fc (B) for 0, 2, 5, 15, 60 or 120 min as indicated. Cells were harvested and cell extracts prepared. Cell extracts were analyzed by Western blot using either Anti-active pAb (upper row) or with corresponding antibodies that recognize both active and inactive forms of each subfamily of MAPK. Results are representative of three experiments with similar results. (C) Activated JNK was detected by ELISA. CD4+ T cells were cultured in precoated 96-well plates at a concentration of 1 × 106 /ml. Cells were stimulated with α-CD3/B7-2 Fc and α-CD3/B7-H2 Fc for different times (1, 5, 15, 60 and 120 min). Following stimulation the cells were fixed. The phosphorylated form of JNK as well as the amount of total JNK was detected using an ELISA method. Results shown are representative for three independent experiments.