Biologic effects of Stat5 proteins on neoplastic mast cell growth. (A) Schematic representation of TAT-Stat5 proteins. DBD indicates DNA binding domain; SH2, Src-homology domain 2; TAD, transactivation domain. (B) Purity of recombinant TAT-Stat5 proteins was assessed by Coomassie blue staining on SDS-PAGE. L indicates bacterial lysate; E, eluate fraction. (C) HMC-1 cells were transduced with the different TAT-Stat5 proteins (10 nM) during 24 hours. Lysates from transduced cS5^F-BM cells were prepared and analyzed by Western blotting with the indicated antibodies. (D) HMC-1 cells were transduced or not with 10 nM of the different TAT-Stat5 proteins during 9 days and the number of living cells was determined every 3 days using the trypan blue dye assay. Results are the mean of 3 independent experiments. (E) HMC-1 cells were transduced with recombinant lentiviruses expressing a Stat5 shRNA or a luciferase shRNA as control. GFP⁺ cells were sorted by flow cytometry and cultured in normal medium for 9 days. Cell extracts were then prepared and analyzed by Western blot with indicated antibodies. (F) The number of viable GFP⁺ cells expressing Stat5 or luciferase shRNAs was also enumerated every 3 days. Representatives of 2 independent experiments are shown.