Impaired GAF and ISGF-3 DNA binding in the cells of P1 early in stimulation. EBV-B cells from WT/WT, P1, K201N/WT, WT/−, P696S/P696S, and −/− subjects were stimulated with the indicated doses of IFN-γ (A) or IFN-α (B) for 15 minutes. Nuclear proteins were extracted to determine GAF (A,C) and ISGF-3 (B,D) levels, with the corresponding probe. Kinetics of GAF (C,E-F) or ISGF-3 (D) DNA-binding activity after IFN-γ and IFN-α, respectively, as determined with stimulation for the times indicated. (F) Pulse-chase experiment with the addition of staurosporine after 15 minutes of IFN-γ stimulation. The results are representative of 3 independent experiments. (G) Schematic diagram of GAF DNA-binding kinetics in healthy control, P1, P696/P696S, and 1928insA/1928insA cells. The x-axis indicates duration of IFN-γ stimulation; and the y-axis, GAF DNA-binding activities.