Effects of SKI-606 treatment on Bcr-Abl and Src kinase activity in CML CD34⁺cells. CML CD34⁺ cells were incubated with SKI-606 or imatinib for 2 hours and 16 hours in low GF containing medium as described in “Western blot analysis.” Protein extracts were prepared as described in “Western blot analysis.” (A) Representative results of Western blotting using anti-CrkL and antiactin antibodies. Densitometry analysis was performed and the phosphorylated CrkL/total CrkL ratio was calculated. (B) Results represent mean plus or minus SEM percentage of phosphorylated CrkL normalized to CrkL phosphorylation in the absence of inhibitors after a 16-hour incubation period based on replicate experiments (n = 4). (C) Representative results of Western blotting using anti-pSrc, anti-Src, and antiactin antibodies. The ratio of phosphorylated Src: total Src was calculated. (D) Results represent mean plus or minus SEM percentage of phosphorylated Src after a 16-hour incubation period normalized to Src phosphorylation in the absence of inhibitors based on replicate experiments (n = 4). (B,D) Significant differences for treated cells compared with untreated controls are indicated for imatinib (†††P < .001; ††P < .01) and SKI-606 (***P < .001; *P < .05). Error bars represent SEM.