Role of PP2A/PP1 in the rapamycin-mediated down-regulation of JNK-1 activity and IL-2 mRNA level.

Peripheral blood T cells were stimulated for 18 hours with either medium alone (lane 1), PMA/αCD28 in the presence (lane 3) or absence of rapamyin (lane 2), PMA/αCD28 in the presence of rapamycin plus okadaic acid (OA; lane 4), or PMAS/αCD28 in the presence of OA (lane 5). After stimulation, cells were divided into 2 groups: one half was used for cellular lysate preparation, and the other half was used to isolate total RNAs. (A) Equal amounts of cellular lysates (35 μg) were used in the immunocomplex kinase assays for JNK-1 using recombinant GST-c-Jun (1-169) as substrate. (B) Equal amounts of RNAs (10 μg) were analyzed by Northern blot analysis using a fragment of IL-2 cDNA as a probe. (C) Graphical representation of the IL-2 mRNA levels quantitated and normalized with 18S ribosomal RNA.