G372C mutation is associated with abnormal phosphorylation and DNA binding activity. (A) Flow cytometric analysis of STAT1 phosphorylation (Y701) of T cells (CD3⁺) after treatment with IFN-α (40 U/μL) or medium alone for 20 minutes at 37°C. Mean fluorescence intensity is indicated. The results are representative of 4 independent experiments. (B) EBV-transformed B cells derived from the patient and a control subject were cultured for 30 minutes with IFN-γ (100 ng/mL), IFN-β (100 ng/mL), or medium alone to analyze STAT1 and STAT3 activation by Western blot. Western blot was carried out with an antibody against Tyr701-phosphorylated STAT1, Tyr705 phosphorylated STAT3, STAT1, STAT3, and actin as a reference. (C-D) DNA-binding activity was analyzed by the electromobility shift activity assay on nuclear extract and carried out in the presence of the ³²P-labeled oligonucleotide STAT-binding probe derived from the IFN-γ response region (GRR) and type I ISRE. EBV-transformed B cells from patient and a healthy subject were stimulated with the indicated doses of IFN-α and IFN-γ for 30 minutes. For the GRR and ISRE probe, the top panel and the bottom panel show 2 exposures for short and long times, respectively. (C) The U3C cell line was transfected with mock, STAT1 wt, and STAT1Δ3 mutant alleles and stimulated with the indicated doses of IFN-α and IFN-γ for 30 minutes. For the GRR and ISRE probe, the top panel and the bottom panel show 2 exposures for short and long times, respectively (D).