Molecular characterization of the GP130 mutants. (A) STAT3 activity, as assessed in a luciferase assay. GP130-deficient HEK293T cells transfected with an empty pCMV6 vector (EV) or with a plasmid encoding the WT or the indicated GP130 mutant plus a pGL4.47 reporter plasmid carrying the luciferase cDNA downstream from five SIEs. Cells were stimulated with the indicated cytokine (+), 24 h after transfection, or were left unstimulated (−) for another 24 h before the measurement of luciferase activity. The horizontal dotted line indicates the luciferase activity after the stimulation, with the indicated cytokine, of cells transfected with the empty pCMV6 vector. The results shown are the mean and standard error of the mean for a technical duplicate. A luciferase assay representative of two independent experiments is shown. (B) STAT3 activity, as assessed in a luciferase assay, after IFN-α stimulation. GP130-deficient HEK293T cells were transfected as described in A. Cells were stimulated with IFN-α (+), 24 h after transfection, or were left unstimulated (−) for another 24 h before the measurement of luciferase activity. The horizontal dotted line indicates the luciferase activity after the stimulation, with the indicated cytokine, of cells transfected with the empty pCMV6 vector. The results shown are the mean and standard error of the mean for a technical duplicate. A luciferase assay representative of two or three independent experiments is shown. (C) GP130-deficient HEK293T cells were transfected with an empty pCMV6 plasmid (EV) or with pCMV6 plasmids encoding the WT or I719fs, S754*, and T761fs GP130 mutants. After 24 h of incubation, the cells were starved of serum for 16 h and then stimulated for 15 min with the indicated GP130-dependent cytokines or left unstimulated, and the phosphorylation of ERK1/2 (pT202/pY204; top panel) or STAT3 (Y705, positive control; lower panel) was evaluated by flow cytometry. Representative results from two independent experiments are shown.