IGF-1R DN increases the activation of IFN-γ/STAT1 pathway in T cells. (A) ST4-E (○), ST4-WT (□), and ST4-DN (▪) cells were cultured in complete medium without (UN) or with IFN-γ (1000 U/mL) for the indicated time intervals. STAT1 activation was evaluated by Western blot analysis of protein cell extracts with an anti–phospho-Tyr (701)–STAT1 Ab. Membranes were subsequently probed with an anti-STAT1 Ab, and the fold induction of IFN-γ–dependent STAT1 phosphorylation was calculated as the ratio between the band intensities of IFN-γ–treated and untreated cells, quantitated after normalization with total STAT1. The fold induction values are shown as means ± SEM from 3 independent experiments at each time point. (B) Expression levels of IRF-1 were evaluated by Western blot on total lysates from ST4-E (○), ST4-WT (□), and ST4-DN (▪) cells treated with or without IFN-γ for 4 and 24 hours. IRF-1 induction, shown as means ± SEM from 3 independent experiments, was quantitated after normalization to actin expression. (C) Western blot evaluation of STAT3 phosphorylation in ST4-E (○), ST4-WT (□), and ST4-DN (▪) cells cultured in complete medium without (UN) or with IFN-γ for the indicated time intervals or with IFN-α for 15 minutes. Membranes were probed with anti–phospho-Tyr (705)–STAT3 and subsequently with anti-STAT3 Abs. Phospho-STAT3 induction, shown as means of fold induction ± SEM from 3 independent experiments, was quantitated as described after normalization to total STAT3 expression.