Characterization of SFK members in AML cells. (A) The levels of Fyn, Lck, Hck, Fgr, and Lyn from normal CD34⁺ HPCs and AML samples were analyzed by Western blot using specific antibodies (patient nos. 1, 2, 4–7, 9, 10, 12–16, 18, 19, 21, 22, 24–28, 30, 31, 33–41). The level of β-actin was assessed as loading control (lower panel). Results shown are representative of the different patients analyzed. (B) Protein lysates from KG1 cells and from AML samples (patient nos. 18, 40, 43, 48) were immunoprecipitated using an anti-Lyn antibody. The immunoprecipitates (IP) were probed with anti-pSrc Y416. The membranes were then stripped and reprobed with a biotinylated anti-Lyn antibody revealed with streptavidin-HRP. Control immunoprecipitates were performed with protein A beads only. (C) KG1, U937, and fresh AML samples (patient nos. 7, 15, 18, 45) were cultured after electroporation with Lyn siRNA or control siRNA. The Western blot was performed 48 to 72 hours after electroporation. Results shown are representative of 4 experiments performed with KG1 cells and of the different patients analyzed. (D) Immunolocalization of Lyn and active forms of SFKs was performed by confocal microscopy using specific antibodies in normal CD34⁺ HPCs and AML samples. Results shown are representative of 3 (for HPC, KG1, and U937) and 8 AML samples (patient nos. 8, 16, 21, 30, 40, 43, 47, 48) independent experiments (original magnification, ×63). Bars represent 5 μm.