Phosphorylation and DNA binding of IFN-activated STAT1^FLAG in BMMΦs.

<p>BMMΦs were isolated from WT and <i>Stat1^ind</i> mice. Cells were pretreated with 0.25 μg/ml dox for 24 h and stimulated with 100 U/ml IFNβ or IFNγ for 15 or 60 min or left untreated. Results are shown from one representative out of three independent experiments. <b>A</b>) Protein lysates were subjected to Western blot analysis for tyrosine 701-phosphorylated STAT1 (P-Tyr-STAT1; upper panel) or serine 727-phosphorylated STAT1 (P-Ser-STAT1; lower panel). Membranes were reprobed with antibodies against STAT1 and FLAG. Loading was controlled by reprobing the membranes with β-tubulin. <b>B</b>) EMSAs were performed to analyze binding of ISGF3 to ISRE (upper panel) or STAT1/STAT1 to GAS elements (lower panel).