FLT3-ITD–mediated STAT5 activation in cells expressing Tyr→Phe mutants. (A) A schematic diagram showing the 5 identified tyrosine-phosphorylated sites and their localization within the FLT3 receptor. The duplicated residues of the N51 internal tandem duplication are also shown. The various Tyr→Phe substitutions created in FLT3-ITD are indicated. ECD indicates extracellular domain; TM, transmembrane domain; TK1 and TK2, tyrosine kinase domains; KI, kinase insert; ITD, internal tandem duplication; and CT, C-terminal tail. STAT5 activation was examined by immunoblot analysis of Ba/F3 (B) or 32D (C) cell lysates starved of IL-3 for 4 hours. Total cell lysates were immunoprecipitated with an anti-STAT5 antibody and separated by SDS-PAGE. Immunoblot analysis was conducted with an antiphosphotyrosine (4G10) antibody (top rows). The blots were then stripped and reprobed with an anti-STAT5 antibody (bottom rows). (D) Pim-1 expression was examined by immunoblot analysis of 32D cell lysates starved of IL-3 for 4 hours. The blot was stripped and reprobed with anti-GAPDH antibody to show equivalent protein loading. Pim-1 is expressed as both a 33 and 44 kDa form in mice due to the presence of an upstream alternative translational start site.²³