Fig. 2: SOCS1 mutations result in uncontrolled STAT pathways activation.

a, b Left: Western blots (WB) of patients (A1, B2, and D1) and healthy controls (CT) derived EBV-B cells stimulated with IFN-γ (10³IU/ml for 1 h) (a) or IL-2 (10⁴IU/ml for 2 h) (b). Lysates were incubated with an antibody against tyrosine-phosphorylated STAT (P-STAT) or against total STAT, as indicated. Right: densitometric quantification of the phospho-STAT/α-tubulin or β-actin ratio upon stimulation. a, b Data are representative of n = 3 (patients B2 and D1), n = 4 (A1, IL-2 stimulation), and n = 6 (A1, IFNγ stimulation) independent experiments. Statistics (versus CT2): IFNγ stimulation, A1 p = 0.0008, B1 p = 0.0029, D1 p = 0.0002; IL-2 stimulation, A1 p < 0.0001, B1 p = 0.0118, D1 p < 0.0001. c The nuclear and cytoplasmic fractions of EBV-B cells from a control (CT) and from patient A1 after stimulation with IFN-γ for 1 h (left) or with IL-2 for 2 h (right) were tested by WB for the presence of P-STAT1 and P-STAT5, respectively. Anti-lamin A/C and anti-α-tubulin antibodies were used to normalize the amount of nuclear and cytoplasmic proteins. Data are representative of two independent experiments. d Real-time quantitative RT-PCR assays of CXCL9 and CXCL10 expression 6 h after stimulation with IFN-γ (left), and assays of CISH and PIM1 expression 6 h after stimulation with IL-2 (right) in EBV-B cells from a CT and from patient A1. Results represent the fold-increased expression between stimulated and unstimulated states and are normalized to endogeneous GAPDH. Data are representative of n = 3 (IL-2 stimulation) and n = 4 (IFNγ stimulation) independent experiments performed in triplicate. Statistics: IFNγ stimulation, CXCL9 p = 0.0094, CXCL10 p = 0.0063; IL-2 stimulation, PIM1 p = 0.0105, CISH p = 0.0008. e Firefly luciferase activity in HEK293T cells transiently transfected with a gamma-activated sequence-driven IFN-γ reporter plasmid (GAS) and expression plasmids for WT or mutant SOCS1 proteins, and then stimulated with IFN-γ for 24 h. The results correspond to the fold-difference between the stimulated state and the unstimulated state. Results represent at least n = 4 independent experiments. All constructs were compared with WT SOCS1. Protein expression from the transfected plasmids was confirmed by immunoblotting the cell lysates (below, one representative result). Statistics: EV p < 0.0001, P123R p < 0.0001, A9FS*76 p < 0.0001, M161FS*46 p < 0.0001, R22W p = 0.0011, Y154H p = 0.0009. a, b, d, e Two-tailed p values were determined in an unpaired t est. Data indicate mean with SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.