<i>Toxoplasma</i> synergizes with IFNγ in terms of phosphorylation and in vitro binding activity of STAT1.

<p>BMDC were left in medium alone (Med), infected with <i>Toxoplasma</i> (Tg, RH strain) at a ratio of 3 parasites per cell, treated with IFNγ (100 ng/ml), or infected with <i>Toxoplasma</i> followed 2 hours later by IFNγ treatment for the indicated times. In (A), cells were fractionated into cytosolic (C) and nuclear (N) extracts prior to immunoblot analysis for phospho-Tyr701-STAT1 (pY-STAT1). PARP and Rab5a served as loading controls for nuclear and cytoplasmic fractions, respectively. In (B) and (C), nuclear extracts were tested for binding to biotinylated probes containing the gamma-activated sequence (GAS) from the <i>Irf-1</i> promoter by EMSA. Supershift assays were also performed with samples in (C) using an antibody against STAT1α or normal rabbit IgG as a negative control. Experiments were repeated at least three times with similar results. GAF, gamma-activated factor (STAT1 homodimer).