Pim-1 protein expression decreases in response to FLT3 inhibition. EOL-1, TF1/ITD, and BaF3/ITD cells were incubated with 50 nM CEP-701 or CEP-5214 for 0 to 6 hours or incubated with an increasing concentration of the inhibitors for 2 hours. Cell extracts were immunoprecipitated with anti-Pim-1 antibody followed by immunoblotting with the same antibody. (A) Time course of treatment with 50 nM CEP-701 or CEP-5214 in EOL-1, TF1/ITD, and TF1 (treated with GM-CSF) cells. (B) Dose response to increasing concentrations of CEP-701 or CEP-5214 treatment of EOL-1 and TF1/ITD cells for 2 hours. (C) Changes in Pim-1 expression, FLT3 phosphorylation, and expression in BaF3/ITD cells in response to treatment with 50 nM CEP-701 for increasing times. Note that mouse Pim-1 also expresses a 44-kDa isoform. (D) Pim-1 expression in BaF3 cells treated with IL-3 plus 50 nM CEP-701 for increasing times.