Constitutive activating phosphorylation of SFKs in leukemic bulk and in immature CD34⁺CD38⁻CD123⁺ leukemic cells. (A) Normal CD34⁺ HPCs unstimulated or stimulated with GM-CSF, SCF and IL-3, KG1, and 53 AML samples (patient nos. 1, 2, 4–22, 24–28, 30, 31, 33–37, 40–44, 47–53, 56–63) were analyzed by Western blot using the phosphospecific antibody recognizing the activated form of SFK members (anti-pSrc Y416). The membranes were stripped and reprobed with the pan total Src antibody and a β-actin antibody as loading control. Data shown are representative of 3 independent experiments. Black stars indicate the position of Lyn. (B) Flow cytometric analysis of a sample from patient 66 using the 4-color protocol as described in “Flow cytometric analysis.” The left panel shows the CD34 versus CD38 dot plot (CD34⁺CD38⁻ blast cell population is represented in gate A). Middle panel shows the CD123⁺ and p-Src Y416 expression in gate A. The right panel shows a sample of the leukemic bulk from patient 66 analyzed both by flow cytometry and by Western blot using the anti-pSrc Y416 antibody. (C) Patient cells were cultured for 1 hour alone or in the presence of PP2 (10 μM), then processed using 4-color immunostaining. The effect of PP2 on SFK phosphorylation was analyzed in the CD34⁺CD38⁻CD123⁺ blast subcompartment (patient nos. 64–68) and in the leukemic bulk (patient nos. 20, 42–44, 47–50, 52–53, 61–63). Results shown are representative of the different patients analyzed.