Figure 2

Phosphorylation of Ser779/780 is required for STAT5A-induced human HS/Pc growth. (a) Schematic representation of recombinant TAT-STAT5A protein and carboxyl-terminal deletion mutants. (b) Human CD34⁺ cells transduced with wild-type or mutant TAT-STAT5A proteins were cultured in presence of SCF for 10 days and the number of viable cells was counted (n=3, **P<0.01). (c) Sequence alignment of human and murine STAT5A/5B isoforms between aa 774 and aa 785 (STAT5A). (d) STAT5A is constitutively phosphorylated on Ser⁷⁸⁰ in human CD34⁺ cells. CD34⁺ cell extracts were analyzed by western blot with an anti-P-Ser⁷⁷⁹⁷⁸⁰-STAT5A antibody (n=3). (e) Human CD34⁺ cell extracts transduced or not (phosphate-buffered saline) with recombinant TAT-STAT5A and TAT-STAT5A^S779A proteins (10 nM) for 24 h were analyzed by western blot with an anti-HA antibody. (f) Growth kinetics of CD34⁺ cells transduced with recombinant TAT-STAT5A or TAT-STAT5A^S779A proteins (n=3; ***P<0.001). DBD, DNA binding domain; SH₂, Src-Homology Domain 2; TAD, transactivation domain.