STAT5b is phosphorylated and localized in the nucleus leading to increased expression of STAT5 target genes in patients with Y665F mutation. (A) MNC samples from LGL patients 1 and 2 (Table 1) carrying the Y665F STAT5b mutation, and a healthy donor were fractionated. Normalized aliquots of total cell (T), cytosolic (C), and nuclear (N) fractions were separated on an SDS-PAGE gel, transferred to PVDF membrane, and western blot analysis was performed using anti-pSTAT5, anti-STAT5 anti-GAPDH (protein loading control), and anti-Histone H3 antibodies (nuclear localization control). (B) Phospho-STAT5 and STAT5 expression were analyzed by ELISA using PB MNC samples from 2 LGL leukemia patients with STAT3 mutation (Y640F and D661V), 2 patients with STAT5b Y665F mutation, 4 patients without STAT3/STAT5b mutation, and 4 healthy controls. (C) A dendrogram showing the clustering analysis of LGL leukemia patients and healthy controls based on the gene expression profile. (D) A heatmap reflecting gene expression profile of 2 LGL leukemia patients with STAT5b mutation Y665F, 1 patient with STAT5b mutation N642H, 3 patients with STAT3 mutation, 2 patients with STAT3/STAT5b WT, and 4 healthy controls. Shown are 29 upregulated genes (log₂ scale) based on the comparison between STAT5b Y665F mutated patients and healthy controls (CD8⁺ cells). On the right, data from 5 known STAT5b target genes are displayed.