Ser585 phosphorylation is selectively deregulated in AML. MNCs from either healthy donors or patients with AML were stimulated with the indicated concentrations of GM-CSF for 5 minutes. The cells were then lysed, and βc was immunoprecipitated with βc-specific mAbs. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with affinity-purified phospho-specific anti–phospho-βcSer585 and anti–phospho-βcTyr577 antibodies. Typical results for a healthy donor and 2 AML samples are shown in panel A and panel B. The Western blot results from GM-CSF dose-response experiments on healthy donors (n = 4) and patients with AML (n = 23) were quantified by laser densitometry, and the quantified signals were converted to a heat map with the use of MeV (www.tm4.org/mev.html), where the intensity of red indicates the magnitude (%) of phosphorylation; gray, samples not done (ND); □, patients who did not show statistically significant constitutive Ser585 phosphorylation; †, samples derived from relapsed patients. Hierarchical cluster analysis was performed for Ser585 phosphorylation signals by using a Euclidean matrix with complete linkage, and the resultant heat maps are shown (C). FAB classifications, cytogenetics, blast count, and percentage of basal phosphorylation of βcSer585 for each AML are indicated alongside the heat maps. (D) Quantitative RT-PCR for the indicated genes was performed on total RNA extracted from purified bone marrow–derived CD34⁺ progenitor cells (n = 4; open boxes), mature CD14⁺ monocytes (n = 4; vertical striped boxes), and AML MNCs (n = 10; AML32-41, horizontal striped boxes). Boxes represent the interquartile range that contains 50% of the values, the horizontal lines mark the median, and the error bars indicate the SD. Data are normalized to β-actin expression and are presented as relative mRNA expression (log₁₀). N/D denotes quantitative RT-PCR not done in bone marrow–derived CD34⁺ progenitor cells.