Immunoblotting.

Exponentially growing Molm14, HB119, or Ba/F3 cells stably expressing mutant isoforms were plated in RPMI medium 1640 + 10% (vol/vol) FCS supplemented with crenolanib at the indicated concentration. HMC1.2 cells were cultured and treated in IMDM + 10% (vol/vol) FCS. After a 90-min incubation, the cells were washed in PBS and lysed and processed as previously described (16). Immunoblotting was performed using anti–phospho-FLT3, anti–phospho-KIT, anti–phospho-STAT5, anti-STAT5, anti–phospho-ERK, anti-ERK, anti–phospho-S6, anti-S6, anti-KIT (Cell Signaling), and anti-FLT3 S18 antibody (Santa Cruz Biotechnology).