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Evaluation of expression and phosphorylation of GOF-STAT1 mutants by using immunobiotting. GOF-STAT1 mutations led to enhanced phosphorylated STAT1 (pSTAT1) expression after stimulation with IFN-γ. U3C cells were transfected with STAT1 mutants or WT and stimulated with IFN-γ for 20 minutes. Western blotting was performed with anti-phosphorylated STAT1, anti-STAT1, and anti-β-actin antibodies. Mutations affecting Y701 prevent STAT1 phosphorylation.
