Fig. 8
From: Structural and functional consequences of the STAT5BN642H driver mutation
In vitro and in cellulo STAT5B/STAT5BN642H phosphorylation and dephosphorylation. a SPR analysis of immobilized His-ABL1 kinase with increasing concentrations of either STAT5B or STAT5BN642H. The binding profile was fit to a two-state model with and the appropriate rate constants are shown. b Ba/F3 cells stably expressing human STAT5B, STAT5BN642H, STAT5BY665F, or empty vector (pMSCV) were starved of IL-3 for 12 h and were then stimulated with 10 ng mL−1 IL-3 for 30 min. Activated, tyrosine phosphorylated STAT5 (pYSTAT5) and total STAT5 levels were analyzed by immunoblotting, and detection of HSC70 was performed as a loading control. c To assess phosphorylation kinetics, Ba/F3 stable cell lines described above were starved of IL-3 for 12 h (no IL-3) and were then stimulated with 10 ng mL−1 IL-3 for 30 min (0 h). Cells were subsequently washed to remove IL-3 and collected at various time points (0.5–12 h). pYSTAT5 and total STAT5 levels were analyzed by immunoblotting, and detection of α-tubulin was performed as a loading control. Blots are representative of two independent experiments. Source data are provided as a Source Data file
