Effects of SKI-606 treatment on MAPK, Akt, STAT5, MEK1/2, and Raf signaling pathways in CML CD34⁺ cells. CML CD34⁺ cells were incubated with SKI-606 or imatinib for 2 hours and 16 hours as described in “Western blot analysis.” Cell lysates were prepared and Western blotting was performed as indicated in the figure. Results of Western blotting for phospho-MAPK, MAPK, phospho-MEK1/2, MEK1/2, phospho-Raf, Raf, and actin after 2 hours (A) and 16 hours (B) of SKI-606 and imatinib treatment, respectively. (C) Results of densitometric analysis of the ratio of phosphorylated MAPK to total MAPK. Results are presented as fold change after inhibitor treatment relative to untreated controls. Significant differences in phosphorylated MAPK expression levels for treated cells compared with untreated controls are indicated for imatinib (†P < .05) and SKI-606 (*P < .05). Results of Western blotting for phospho-Akt, Akt, phospho-STAT5, STAT5, and actin after 2 hours (D) and 16 hours (E) of SKI-606 and imatinib treatment, respectively. Results shown represent mean plus or minus SEM fold increase of phosphorylated protein compared with the untreated control based on replicate experiments (n = 5).