cS5 confers factor-independent survival in the presence of intact serine phosphorylation only. (A) Transduced and FACS-sorted Ba/F3 cells were analyzed for GFP expression by flow cytometry and showed a consistently high percentage of GFP⁺ cells. (B) Representative cytospins of IL-3–dependent Ba/F3 cells transduced with the indicated Stat5a variants and kept in medium with/without addition of IL-3. After 7 days, all transduced cells proliferated in the presence of cytokine. However, only cS5-transduced Ba/F3 cells were repeatedly able to grow independent of IL-3. Cells transduced with WT Stat5a, cS5-S725A, cS5-S779A, and cS5-SASA were not transformed to factor independence and underwent apoptosis similar to untransduced Ba/F3 controls without IL-3. (C) Growth factor–dependent Ba/F3 cells were transduced with the different cS5 constructs and WT Stat5a as indicated and FACS-sorted for GFP⁺ cells. Upon IL-3 withdrawal, apoptosis was analyzed by FACS using an annexin V antibody at 72 hours and at 7 days. cS5-expressing cells were the only ones able to survive without cytokine stimulation. All other mutants underwent apoptosis after factor depletion in 3 individual experiments. (D) Cell lysates from sorted GFP⁺ cells were prepared and analyzed by immunoblotting with the indicated antibodies. Parental Ba/F3 cells showed weak expression of total Stat5, which was strongly tyrosine phosphorylated upon IL-3 stimulation. In all Ba/F3 cells transduced with the different cS5 constructs, Stat5 was highly tyrosine phosphorylated in the absence of cytokine stimulation.