Stable transfection of Ba/F3

To establish Ba/F3 cells stably expressing FLT3-WT and FLT3-ITD, EcoPack packaging cells were transfected with pMSCVpuro-FLT3-WT or pMSCVpuro-FLT3-ITD construct, and virus-containing supernatants were collected 72 h after transfection as described previously [40, 41]. Retroviral infection of Ba/F3 cells was followed by a 2-week selection in 1.2 μg/ml puromycin. FLT3 expression was confirmed by flow cytometry and western blotting. Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD cells were then further infected with virus produced in EcoPack cells by transfecting with pMSCVneo-FYN-Myc-FLAG construct or empty vector. Three days after infection cells were selected with 0.8 mg/ml G-418 for 2 weeks, and FYN expression was verified by western blotting.