Downstream molecular mechanisms of CD34⁺ cells expressing high and low levels of Bcr-Abl. CD34⁺GFP⁺ cells were grown in SFM containing low GF for 7 days followed by preparation of total cell lysates for Western blotting and nuclear extracts for EMSA assays. (A) Results of Western blotting for total and phosphorylated AKT (n= 4), MAPK (n= 4), and STAT5 (n= 5) in control and Bcr-Abl–expressing cells. Representative blots are shown on the left, and cumulative results from densitometric analysis of multiple experiments are shown in the graphs on the right. Significance level comparing BA^lo and BA^hi cells is *P < .014. (B) We further analyzed the promoter-binding activity of STAT5 using electromobility shift assay (EMSA) (lanes 1-3). Specificity of binding is shown by blockage by addition of excess unlabeled probe to the binding reaction (lanes 4-6). The presence of STAT5 protein within the complex is shown by supershift following addition of anti-STAT5 antibodies to the binding reaction (lanes 7-9). (C) Western blot analysis of expression of antiapoptotic proteins Bcl-X_S/L, Mcl-1, and Bcl-2. Representative blots are shown on the left and cumulative results from densitometric analysis of multiple experiments are shown in the graphs on the right.