Figure 5.

p21^Cip1 preferentially interacted with phosphorylated c-Jun N-terminla kinase (p-JNK) and p-c-jun in anergic T helper type 1 (Th1) cells. (a) The Th1 cells were stimulated with anti-CD3 and anti-CD28 antibody-coated beads for 0–24 hr, lysed at different time-points, separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)and immunoblotted with antibody specific for p21^Cip1. (b) The Th1 cells were stimulated with antigen in the presence or absence of n-butyrate in primary cultures. Viable control or anergic Th1 cells were then isolated. Control Th1 cells were stimulated for 24 hr with anti-CD3 and anti-CD28 antibody-coated beads (lane 1). febs_Half of the anergic Th1 cells were lysed at the end of primary cultures (lane 2) and the other half were restimulated with anti-CD3 and anti-CD28 antibody-coated beads for 2 hr before lysis (lane 3). Cell lysates were prepared from the three different groups and p21^Cip1 was immunoprecipitated from the lysates. The immunoprecipitated p21^cip1 as well as the remaining lysate supernatant (SN) were separated on SDS–PAGE and then immunoblotted with antibodies specific for p21^Cip1, cdk2, PCNA, JNK, p38, p-JNK and p-c-jun. This experiment was repeated once with similar results.