The tyrosine phosphorylation and DNA binding of STAT5 in response to IL-12 is enhanced in T cells activated with PHA + IFN-γ but inhibited in T cells activated with PHA + IL-4. (A) PBMC cultured in either PHA alone, PHA + IFN-γ, or PHA + IL-4 were stimulated with medium alone (lanes 1, 4, and 7), IL-12 (lanes 2, 5, and 8), or IL-2 (lanes 3, 6, and 9). Western blots were performed with antibodies to phospho-STAT5 (upper panel) or STAT5 (lower panel). Results are representative of five separate experiments. (B) PBMC cultured in either PHA alone or PHA + IFN-γ were stimulated with medium alone (lanes 1 and 4), IL-12 (lanes 2 and 5), or IL-2 (lanes 3 and 6). Cell lysates were immunoprecipitated with a STAT5 antibody, and Western blots were performed with antibodies to phosphotyrosine (upper panel), phospho-STAT5 (middle panel), or STAT5 (lower panel). Results are representative of three separate experiments. (C) PBMC were activated with PHA alone, PHA + IFN-γ, or PHA + IL-4 and then stimulated with the indicated cytokine. Nuclear lysates were then prepared and used in an EMSA with a³²P-labeled DNA probe consisting of a STAT-binding sequence found within the IRF-1 gene promoter. Where indicated, 1 μL of a STAT5 antibody was added to the binding reaction containing nuclear lysate and DNA probe. The arrow points to supershifted complexes. Similar results were obtained in two separate experiments.