American Society of Hematology

Figure 1.

$Figure 1. CEP-701 inhibits FLT3 autophosphorylation reversibly and induces cytotoxicity in EOL-1 cells. (A) EOL-1 cells were incubated with increasing concentrations of CEP-701. The graph shows cytotoxicity assessed by MTT assay after 48 hours. The inset shows whole-cell extracts (500 μg/sample) that were prepared after 1 hour of exposure to CEP-701 and immunoprecipitated with anti-human FLT3 antibody, followed by separation in 8% polyacrylamide gel electrophoresis and immunoblotting with antiphosphotyrosine antibody. The membrane was stripped and reprobed with anti-FLT3 antibody to demonstrate equal loading of FLT3 in each lane. (B) Aliquots of EOL-1 cells were incubated with 50 nM CEP-701 for 4 hours and 8 hours. Additional cell aliquots were then washed by PBS to remove the inhibitor and incubated for an additional 4 and 8 hours. Most of the cells from each of these time points were harvested for RNA preparation for microarray analysis. A small fraction was harvested for analysis of FLT3 phosphorylation status as shown.$

CEP-701 inhibits FLT3 autophosphorylation reversibly and induces cytotoxicity in EOL-1 cells. (A) EOL-1 cells were incubated with increasing concentrations of CEP-701. The graph shows cytotoxicity assessed by MTT assay after 48 hours. The inset shows whole-cell extracts (500 μg/sample) that were prepared after 1 hour of exposure to CEP-701 and immunoprecipitated with anti-human FLT3 antibody, followed by separation in 8% polyacrylamide gel electrophoresis and immunoblotting with antiphosphotyrosine antibody. The membrane was stripped and reprobed with anti-FLT3 antibody to demonstrate equal loading of FLT3 in each lane. (B) Aliquots of EOL-1 cells were incubated with 50 nM CEP-701 for 4 hours and 8 hours. Additional cell aliquots were then washed by PBS to remove the inhibitor and incubated for an additional 4 and 8 hours. Most of the cells from each of these time points were harvested for RNA preparation for microarray analysis. A small fraction was harvested for analysis of FLT3 phosphorylation status as shown.

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