Frizzled-4 is regulated by activating Flt3 mutations. (A) Bar graph representing the relative Frizzled-4 mRNA expression levels as analyzed by microarray analyses.³⁵  Expression levels in Flt3-WT, either stimulated with FL or left untreated, were set as 1. (B) Quantitation of Frizzled-4 mRNA levels by real-time RT-PCR. The 32D cells stably transfected with either wild-type Flt3 (Flt3-WT) or Flt3-ITD were depleted from IL-3 and cultured for 36 hours in FL (Flt3-WT) or without cytokines (Flt3-ITD). Then, the indicated growth factors were added for an additional 12 hours. Samples were taken for RNA preparation, and real-time RT-PCR analyses were performed. The mean of the Flt3-WT level was set as 1. Each bar represents the mean of 2 independent experiments ± SD. (C) Detection of Frizzled-4 protein levels. 32D/Flt3-WT or 32D/Flt3-ITD cells were cultured for 12 hours in the absence of growth factors. Subsequently, the indicated growth factors were added for 6 hours. Frizzled-4 protein levels were determined by Western blot analyses.