Fig. 1

Secondary mutation increases the ligand-independent activation of KIT. a Wild-type KIT or KIT mutants in pMSCVpuro were transfected into EcoPack cells, supernatant was collected to infect Ba/F3 cells. After selection with puromycin, expression of KIT was examined by flow cytometry. Light gray: isotype control, dark gray: PE-anti-KIT antibody. b Ba/F3 cells stably expressing wild-type KIT or KIT mutants were washed and starved in RPMI 1640 medium for 4 h before stimulation with 100 ng/ml SCF for 2 min, KitC1 was used to precipitate KIT from cell lysate, after separation by SDS-PAGE and transfer to PVDF membrane, pY antibody 4G10 and KitC1 were used to detect KIT activation. pAkt, Akt and β-actin antibody were used to detect Akt activation in total cell lysates. c KIT activation was studied in Ba/F3 cells stably expressing primary and/or secondary KIT mutants as described above. Signal intensity was quantified and calculated to show the relative KIT activation