Role of tyrosines 589 and 591 in FLT3-D835Y–mediated FLT3 activation and STAT5 signaling. (A) STAT5 activation was examined in 32D cells stably expressing FLT3WT, FLT3-ITD (N51), N51-Y589/591F, D835Y, or D835Y-Y589/591F. Cells were starved of IL-3 for 4 hours. Cell lysates were immunoprecipitated with an anti-STAT5 antibody and separated by SDS-PAGE. Immunoblot analysis was conducted with an antiphosphotyrosine (4G10) antibody. The blot was then stripped and reprobed with an anti-STAT5 antibody. (B) FLT3 activation was also analyzed in the same cells by immunoprecipitation with anti-FLT3 antibody followed by immunoblot analysis with an antiphosphotyrosine antibody.