Figure 3.
Effects of mutations on PTPN2 phosphatase activity and JAK-STAT signaling. (A) Tyrosine PTPN2 phosphatase activity of immunoprecipitated WT or mutant PTPN2 toward fluorescent tyrosine-phosphorylated STAT1 or STAT3 peptide by RP-UFLC. Mean and SDs are shown and represent three different experiments (ns, nonsignificant, *P < 0.05, ***P < 0.001, Paired t test). (B) STAT1 transcriptional activity following 24-h activation with IFN-α of HEK293T cells expressing IFN-sensitive responsive element (ISRE) and transfected with the indicated constructs (left panel). Mean and SDs are shown and representative of at least five different experiments in triplicate STAT3 transcriptional activity following 24-h activation with IL-6 of HEK293T cells expressing STAT3 responsive element and transfected with the indicated constructs (right panel). Results were normalized on Renilla. Mean and SDs are shown and representative of at least three different experiments in triplicate (ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test). (C) PTPN2 protein expression in Jurkat–T cells and PTPN2 Crispr KO Jurkat–T cells. PTPN2 expression was quantified using anti-PTPN2 antibody and automated quantitative western blot (left panels). Mean and SDs are shown and representative of three different experiments. PTPN2 constructs were re-expressed in Jurkat–T cells following lentiviral transduction and protein expression was verified using anti-HA antibody and automated quantitative western blot (right panels). Mean and SDs are shown and representative of five different experiments. (D) Modified Jurkat–T cells were stimulated with IFN-α or IFN-γ as indicated and phosphorylation of STAT1 was measured by intracellular staining and analyzed by spectral cytometry. pSTAT1 phosphorylation in Jurkat transduced with PTPN2 WT was used as reference. Histograms representative of one experiment out of four are illustrated on the right of each quantification (ns, nonsignificant, *P < 0.05, **P < 0.01, one-way ANOVA, Kruskal–Wallis test). All MW are in kDa. Source data are available for this figure: SourceData F3.

Effects of mutations on PTPN2 phosphatase activity and JAK-STAT signaling. (A) Tyrosine PTPN2 phosphatase activity of immunoprecipitated WT or mutant PTPN2 toward fluorescent tyrosine-phosphorylated STAT1 or STAT3 peptide by RP-UFLC. Mean and SDs are shown and represent three different experiments (ns, nonsignificant, *P < 0.05, ***P < 0.001, Paired t test). (B) STAT1 transcriptional activity following 24-h activation with IFN-α of HEK293T cells expressing IFN-sensitive responsive element (ISRE) and transfected with the indicated constructs (left panel). Mean and SDs are shown and representative of at least five different experiments in triplicate STAT3 transcriptional activity following 24-h activation with IL-6 of HEK293T cells expressing STAT3 responsive element and transfected with the indicated constructs (right panel). Results were normalized on Renilla. Mean and SDs are shown and representative of at least three different experiments in triplicate (ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test). (C) PTPN2 protein expression in Jurkat–T cells and PTPN2 Crispr KO Jurkat–T cells. PTPN2 expression was quantified using anti-PTPN2 antibody and automated quantitative western blot (left panels). Mean and SDs are shown and representative of three different experiments. PTPN2 constructs were re-expressed in Jurkat–T cells following lentiviral transduction and protein expression was verified using anti-HA antibody and automated quantitative western blot (right panels). Mean and SDs are shown and representative of five different experiments. (D) Modified Jurkat–T cells were stimulated with IFN-α or IFN-γ as indicated and phosphorylation of STAT1 was measured by intracellular staining and analyzed by spectral cytometry. pSTAT1 phosphorylation in Jurkat transduced with PTPN2 WT was used as reference. Histograms representative of one experiment out of four are illustrated on the right of each quantification (ns, nonsignificant, *P < 0.05, **P < 0.01, one-way ANOVA, Kruskal–Wallis test). All MW are in kDa. Source data are available for this figure: SourceData F3.

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