IM inhibits KIT cITD-induced ligand-independent constitutive tyrosine phosphorylation of STAT3 and STAT5 in Ba/F3/kit cITD. Phosphorylation status of the KIT downstream signaling targets was determined by Western blot analysis after inhibition with IM, Rap, and Ly294002. After a 5-hour starvation period, 1 × 10⁷ cells were stimulated for 6 hours in 1 mL medium with or without 250 ng/mL KL and inhibitors at concentrations 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; phospho-Akt (S⁴⁷³) is specific for AKT phosphorylated on serine 473; phospho-p70S6K (T³⁸⁹) is specific for p70S6K phosphorylated on tyrosine 389; phospho-STAT3 (Y⁷⁰⁵) recognizes STAT3 phosphorylated on tyrosine 705; phospho-STAT5 (Y⁶⁹⁴) recognizes STAT5 phosphorylated on tyrosine 694; 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/kit cITD stimulated with 250 ng/mL KL for 15 minutes.