FLT3-JM-PM mutants expressed in Ba/F3 cells show constitutive activation of STAT5 and up-regulation of Bcl-x(L). (A) FLT3-WT, FLT3-W51, FLT3-V592A, FLT3-V579A, FLT3-F594L, FLT3-F590GY591D, or mock-transduced cells were starved for 24 hours in the presence of 0.3% FBS and stimulated with 100 ng FL /mL for 5 minutes. Crude-cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on a nitrocellulose membrane. Blots were incubated with anti-phospho-STAT5 antibody, stripped, and reblotted with anti-STAT5 antibody. (B) FLT3-ITD-expressing cells (W51, NPOS, and W78) and FLT3-TKD-expressing cells (D835Y and D835V) were analyzed as described in panel A, and the films were subjected to densitometric analysis to quantify the percentage of phospho-STAT5 in relation to total STAT5 amount in unstimulated cells. (C) Crude-cell lysates were subjected to Western blot analysis using a monoclonal antibody against Bcl-x(L). Bcl-x(L) overexpressed in Ba/F3 cells served as a positive control. Equal protein loading in all lanes was confirmed by immunoblotting using an anti-β-actin antibody.