IL-12–induced STAT4 tyrosine phosphorylation and DNA binding is not augmented by IFN-γ but is partially inhibited by IL-4. (A) PBMC were cultured with either PHA alone or PHA + IFN-γ and then stimulated with medium alone (lanes 1 and 4), IL-12 (lanes 2 and 5), or IL-2 (lanes 3 and 6). Immunoprecipitations were performed on whole cell lysates with an antibody to STAT4, followed by Western blots with an antiphosphotyrosine antibody (upper panel) or an antibody to STAT4 (lower panel). Results are representative of three separate experiments. (B) PBMC were cultured with either PHA alone or PHA + IL-4 and stimulated with medium alone (lanes 1 and 3) or IL-12 (lanes 2 and 4). STAT4 was then immunoprecipitated from cell lysates as in (A), and Western blots were performed as described in (A). Results are representative of three separate experiments. (C) PBMC were activated with PHA, 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 a 32P-labeled DNA probe consisting of a STAT-binding sequence found within the promoter region of the IRF-1 gene. Where indicated, 1 μL of a STAT4 antibody was added to the binding reaction containing nuclear lysate and DNA probe. Similar results were obtained in two separate experiments.
