STAT pathways rather than MAPK pathways are constitutively activated by both ECD and PTD-KIT mutants. Parental or KIT-expressing Ba/F3 cells were serum starved for 5 hours in medium containing 0.5% FBS before stimulation for 5 minutes with or without 250 ng/mL mSCF as indicated. Ba/F3-WT (PS) cells were cultured in the presence of 250 ng/mL mSCF as a control for permanent stimulation (PS) of the KIT receptor. Whole-cell lysates were analyzed by SDS-PAGE, followed by Western blotting with indicated antibodies. (A) After immunoblotting with phospho-specific antibodies toward STAT1, STAT3, and STAT5 (top panels), membrane was stripped and reprobed with anti-STAT antibodies as loading controls. (B) Blots were stained with the indicated activation-specific antibodies. (Top panels) Antiphospho-ERK1/2, antiactive P38, and antiphospho-JNK antibodies. Blots were stripped and reprobed with anti-ERK2 and anti JNK antibodies to illustrate equal loading. These results are representative of 3 independent experiments.