Sustained Stat4 activation is associated with T-bet induction. (A) BALB/c CD8 T cells were stimulated with anti-CD3 plus anti-CD28 mAbs, in combination with indicated cytokines (10 ng/mL each) for 24 hours, and real-time RT-PCR was used to measure T-bet expression. Data shown are one representative of 3 independent experiments; standard errors of PCR triplicates are shown. (B) Western blotting for Stat4 activation. BALB/c CD8 T cells were stimulated with anti-CD3 plus anti-CD28 mAbs plus the indicated cytokines for 30 minutes. Antiphosphorylated Stat4 or anti-Stat4 Abs were used for Western blotting. (C) IL-12 but not IFNα or IFNβ induces sustained Stat4 activation at 24 hours after stimulation. CD8 T cells were stimulated with anti-CD3 plus anti-CD28 mAbs, with or without IL-12, IFNα, or IFNβ. Data shown are one representative of 3 independent experiments; standard errors of PCR triplicates are shown.