C-terminal truncation mutant of STAT5 (▵STAT5) inhibited STAT5 activation in Ba/F3 cells. (A) Ba/F3 cells were infected with retrovirus encoding STAT5, ▵STAT5, or the virus vector, and GFP-positive cells were sorted by FACS. P shows the GFP-positive cell populations, whereas N shows the GFP-negative cell populations used for the analysis. (B) GFP-positive and -negative cells were examined for the expression of STAT5b and ▵STAT5 by Western blotting. Sorted cells were lysed and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted with anti-STAT5 monoclonal antibody (Transduction Laboratory, Lexington, KY). (C) Effect of ▵STAT5 on the transactivation of β-casein Luciferase reporter. GFP-positive and -negative cells were cultured in the presence of 1 ng/mL IL-3 for 24 hours and subjected to the luciferase assays. The relative Luciferase activity was calculated as the ratio of the Luciferase activity of GFP-positive cells infected with the retrovirus vector stimulated with IL-3. The means ± SEM for three experiments are shown.