Effects of dasatinib on Lyn and Btk activation in neoplastic MCs. (A) Total cell lysates from HMC-1.1 cells (TCL) and proteins that were bound by immobilized dasatinib were immunoblotted for KIT and Btk. Ampicillin served as negative control. (B) HMC-1.1 cells and HMC-1.2 cells were cultured in control medium or in the presence of dasatinib (0.01-1μM for 4 hours). Thereafter, cells were subjected to Western blot analysis using Abs directed against p-Lyn^Tyr507, p-Src^Tyr416, Lyn, p-Btk^Tyr223, Btk, and KIT. The β-actin loading control is also shown. To detect KIT activation, an immunoprecipitation was conducted using an anti-KIT Ab followed by immunoblotting using the anti-phosphotyrosine Ab 4G10. (C) HMC-1.2 cells were incubated in control medium (Co) or in medium containing dasatinib (1μM) for 4 hours or 24 hours at 37°C. Then, cells were spun on cytospin slides and stained with Abs specific for Lyn, p-Lyn^Tyr507, p-Lyn^Tyr396, Btk, and p-Btk^Tyr551 by indirect immunocytochemistry. Cells were analyzed using an Olympus AX-1 microscope equipped with 60×/1.35 UPlan-Apo objective lens. Image acquisition was performed using an Olympus DP11 camera and Adobe Photoshop CS2 software Version 9.0 (Adobe Systems). Magnification, ×600. (D) Primary neoplastic mast cells from one patient with aggressive systemic mastocytosis (ASM) and one with mast cell leukemia, MCL (no. 31 and no. 36 in Table 1) were cultured in control medium or in the presence of midostaurin (1μM) or dasatinib (1μM) for 12 hours. Thereafter, cells were subjected to Western blot analysis using Abs against p-Lyn^Tyr507, p-Src^Tyr416, Lyn, p-Btk^Tyr223, Btk, and β-actin. (E) Immunocytochemistry performed with primary mast cells (ASM patient 30) exposed to control medium or medium containing dasatinib (1μM) for 12 hours. Abs used and the microscopy technique applied were the same as that described for panel C. Magnification, ×600.