K201N mutation does not impair STAT1 dephosphorylation and homodimerization. (A) K201N does not impair homodimerization. U3C cells were transfected with a combination of mock (M), WT, and K201N-mutated STAT1 plasmids tagged with either Flag or Myc. Proteins were extracted 48 hours after transfection and subjected to coimmunoprecipitation and Western blotting. (Bottom panel) Western blotting for the detection of Myc or Flag expression in input with a specific antibody. (Top panel) Immunoprecipitation with an anti-Flag antibody, followed by Western blotting with an anti-Myc or anti-Flag antibody. (B) STAT1 phosphorylation kinetics. EBV-B cells from a healthy control (WT/WT), P1, a subject heterozygous for the K201N allele (K201N/WT), and a patient with complete STAT1 deficiency (1928insA/1928insA, −/−) were stimulated with 105 IFN-γ for the time indicated (in minutes). Western blotting was carried out with an antibody against Tyr701-phosphorylated STAT1 (P-STAT1), or STAT1α (STAT1), or with an antibody against α-tubulin, as a reference. (C) Pulse-chase experiment. EBV-B cells from a healthy control (WT/WT), P1, a subject heterozygous for the K201N allele (K201N/WT), and a patient with complete STAT1 deficiency (1928insA/1928insA, −/−) were stimulated with 105 IU/mL IFN-γ for 30 minutes, and staurosporine was added and the mixture incubated for the time indicated. Western blot shows Tyr701-phosphorylated STAT1 and STAT1, with STAT2 as a reference.
