STAT-1 activation kinetics induced by IFN-γ in malignant T cells. ST4 T cells untreated or treated for 48 hours with IGF-1 (100 ng/mL) or anti–IGF-1R blocking mAb (10 μg/mL) were cultured without (UN) or with IFN-γ (1000 U/mL) for the indicated time intervals. (A) STAT-1 activation was evaluated by Western blot analysis of nuclear cell extracts with anti–phospho-tyr (701)-STAT-1 mAb. Western blot filters were subsequently probed with an anti–STAT-1 antibody to confirm equal protein loading in each lane of the gel. (B) STAT-1 activation was evaluated by EMSA assay of nuclear extracts. Medium, IGF-1, or anti–IGF-1R mAb 48-hour–pretreated cells were incubated in the absence (UN) or in the presence of 1000 U/mL IFN-γ at the appropriate time interval. The experiments were performed independently at least 3 times.