Molecular characterization of SU5614-resistant cells. (A) The juxtamembrane region of FLT3 was amplified by PCR with the use of the published primer pair 11F and 12R,^48,49  and PCR products were separated by agarose gel electrophoresis and viewed under UV illumination after ethidium bromide staining. M indicates DNA molecular weight marker; H₂O, water control; Ba/F3, native Ba/F3 cells; WT, Ba/F3 FLT3-WT; ITD, Ba/F3 FLT3-ITD; ITD-R1-4, Ba/F3 FLT3-ITD-R1-4; PP, positive control (patient with AML carrying a FLT3-LM). (B) Detection of FLT3-TKD mutations was performed by melting curve analysis after amplification of a 244-bp fragment by real-time PCR as described previously.⁵⁰  In the presence of the FLT3-TKD wild-type sequence or the FLT3 TKD mutant DNA the fluorescence peak is observed at 63°C and 55°C, respectively. (C) The structural domains of the FLT3 protein with the position of the TKD mutation found in Ba/F3 FLT3-ITD-R1-4 cells (D835N and Y842H) are shown.