Kinetics of IFN-γ–dependent Stat1 and Stat1K703R DNA binding. (A) Stat1^–/– MEFs, infected with retroviral vectors directing expression of either Stat1 (St1) or Stat1^K703R (St1^K703R), were stimulated with IFN-γ (66 U/mL for 30 minutes). Dissociation kinetics of Stat1 DNA binding activity were determined by incubating the IFN-γ–stimulated extracts with a labeled GAS probe (0.1 pmol for 20 minutes at 22°C) and then chasing with a 100-fold excess of cold probe. Aliquots were removed at indicated times and loaded onto a running gel. (B) Quantification of DNA binding from panel A was determined by ImageQuant (BD Molecular Dynamics, Santa Clara, CA) and plotted as fraction bound versus time. (C) Relative affinity was determined by simultaneously incubating IFN-γ–stimulated WCEs (from panel A) with a labeled GAS probe (0.1 pmol) and increasing molar excess of cold probe, as indicated (20 minutes at 22°C). Samples were then run on EMSA gel. (D) Quantification of DNA binding results from panel C was determined by imagequant and plotted as fraction bound versus molar excess of cold probe. Data are representative of 3 independent studies. Error bars indicate standard deviation.