dbcAMP inhibits tyrosine phosphorylation of STAT5. (A). Primed T cells were cultured for 24 hours in either medium alone or in the presence of 1 mmol/L dbcAMP and then stimulated with IL-2 (1,000 IU/mL) for 15 minutes. Cell lysates were then prepared and immunoprecipitated with anti-STAT5 antibody. Western blotting was performed on aliquots of the immunoprecipitate to determine whether STAT5 was tyrosine phosphorylated (4G10 Ab) and to assess the level of STAT5 in the immunoprecipitates (anti-STAT5 Ab). (B) Aliquots of the same cells were subjected to Western blotting to determine if dbcAMP suppressed JAK3. (C) EMSA and supershift analyses were performed as indicated in Materials and Methods using nuclear extracts from samples shown in (A). The arrow indicates the migration of the IL-2 inducible complex binding to IRF1-GAS sequence. The loss of this inducible band in the presence of anti-STAT5 antibody shows that this band contains STAT5.