Figure 1.
Figure 1. STAT5B mutation characterization. (A) Linear representation of the STAT5B protein structure. Previously known LGL leukemia mutations in STAT5B are marked in the SH2 domain, whereas the novel Q706L and S715F mutations in the transactivation domains are marked with green boxes. Multiple tyrosine and serine phosphorylation sites are marked in red. (B) STAT5B reporter assay results. Mutated STAT5B constructs (pCMV6-XL6 STAT5B) were generated through site-directed mutagenesis followed by transfection and expression of WT and mutated STAT5B (Q706L, S715F, N642H) in HeLa cells together with a STAT5B reporter. Dual-reporter luciferase assay was used to determine activation and phosphorylation of mutated STAT5B. The experiment was repeated 3 times. Columns represent mean of the fold-change activity. Error bars indicate the standard error of the mean (SEM), and the statistical significance was calculated with a 1-way analysis of variance (ANOVA; *P < .05, **P < .001). (C) To investigate the phosphorylation status of the variants, HeLa cells transfected with the abovementioned variants were analyzed by western blot with a phosphoSTAT5 (Tyr694) specific antibody. Protein lysates of the different variants were separated on an sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. STAT5 protein levels of the different variants were used to normalize for the transfection efficacy. β-Actin was used as a loading control. (D) Transfected HeLa cells were stimulated with 100 ng/mL interferon-α for 6 hours. A dual-reporter luciferase assay was used to determine activation and phosphorylation of mutated STAT5B. The experiment was repeated 2 times. Columns represent mean of the fold-change activity. Error bars indicate the SEM, and the statistical significance was calculated with a 1-way ANOVA (*P < .05, **P < .001). (E) Typical morphology of a representative LGL cell in a STAT5B mutated T-LGL patient. Scale bar, 15 μm. (F) Morphology of lymphocyte expressing CD4, CD56, and TCRαβ in a healthy individual. CD4+CD56+ and TCRαβ-type lymphocytes were sorted by the FACS method and stained with Wright-Giemsa stain. A representative cell is shown. Scale bar, 15 μm.

STAT5B mutation characterization. (A) Linear representation of the STAT5B protein structure. Previously known LGL leukemia mutations in STAT5B are marked in the SH2 domain, whereas the novel Q706L and S715F mutations in the transactivation domains are marked with green boxes. Multiple tyrosine and serine phosphorylation sites are marked in red. (B) STAT5B reporter assay results. Mutated STAT5B constructs (pCMV6-XL6 STAT5B) were generated through site-directed mutagenesis followed by transfection and expression of WT and mutated STAT5B (Q706L, S715F, N642H) in HeLa cells together with a STAT5B reporter. Dual-reporter luciferase assay was used to determine activation and phosphorylation of mutated STAT5B. The experiment was repeated 3 times. Columns represent mean of the fold-change activity. Error bars indicate the standard error of the mean (SEM), and the statistical significance was calculated with a 1-way analysis of variance (ANOVA; *P < .05, **P < .001). (C) To investigate the phosphorylation status of the variants, HeLa cells transfected with the abovementioned variants were analyzed by western blot with a phosphoSTAT5 (Tyr694) specific antibody. Protein lysates of the different variants were separated on an sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. STAT5 protein levels of the different variants were used to normalize for the transfection efficacy. β-Actin was used as a loading control. (D) Transfected HeLa cells were stimulated with 100 ng/mL interferon-α for 6 hours. A dual-reporter luciferase assay was used to determine activation and phosphorylation of mutated STAT5B. The experiment was repeated 2 times. Columns represent mean of the fold-change activity. Error bars indicate the SEM, and the statistical significance was calculated with a 1-way ANOVA (*P < .05, **P < .001). (E) Typical morphology of a representative LGL cell in a STAT5B mutated T-LGL patient. Scale bar, 15 μm. (F) Morphology of lymphocyte expressing CD4, CD56, and TCRαβ in a healthy individual. CD4+CD56+ and TCRαβ-type lymphocytes were sorted by the FACS method and stained with Wright-Giemsa stain. A representative cell is shown. Scale bar, 15 μm.

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