Constitutive tyrosine phosphorylation of KIT cITD, and ligand-independent constitutive phosphorylation of the PI3K/AKT/mTOR pathway, STAT1, STAT3, STAT5, and SHP-2. (A) Phosphorylation status of the KIT WT receptor and p85 PI3K with and without KL was determined by Western blot analysis and compared with the 4 KIT cITD variants. After a 5-hour starvation period, 1 × 10⁷ cells were stimulated for 15 minutes in 1 mL medium with or without 250 ng/mL KL as indicated. Whole-cell lysates (20 μg per lane) were subjected to SDS-PAGE and immunoblotted with anti-phospho-specific antibodies as indicated. Phospho-KIT (Y⁷¹⁹) recognizes KIT phosphorylated on Tyr⁷¹⁹; phospho-p85 PI3K recognizes the phosphorylated regulatory subunit p85 of PI3K. (B) Phosphorylation and downstream signaling targets of WT KIT and KIT cITD1 were determined by Western blot analysis. Phospho-Akt (S⁴⁷³) is specific for AKT phosphorylated on serine 473; phospho-p70S6k (T³⁸⁹) is specific for p70S6K phosphorylated on tyrosine 389; phospho-S6RP (S^235/236) recognizes S6RP phosphorylated on serine 235/236; and phospho-ERK 1/2 (T²⁰²/Y²⁰⁴) is specific for the subunits p42 and p44 of the MAP kinases ERK1 and ERK2 phosphorylated on threonine 202 and tyrosine 204. Subsequently, the blots were stripped and stained with the indicated protein-specific antibodies to demonstrate equal loading. Ba/F3 BSD indicates mock-transfected Ba/F3 cells. (C) Phospho-STAT1 (Y⁷⁰¹) recognizes phosphorylated STAT1 on tyrosine 701; phospho-STAT3 (Y⁷⁰⁵) recognizes STAT3 phosphorylated on tyrosine 705; and phospho-STAT5 (Y⁶⁹⁴) recognizes STAT5 phosphorylated on tyrosine 694. Phospho-SHP-2 (Y⁵⁴²) is specific for SHP-2 phosphorylated on tyrosine 542. Subsequently, the blots were stripped and stained with the indicated protein-specific antibodies to demonstrate equal loading. Ba/F3 BSD indicates mock-transfected Ba/F3 cells.