Synergistic drug interactions between midostaurin and dasatinib may be mediated by the Lyn/Btk pathway and downstream STAT5. (A) HMC-1.2 cells were kept in control medium or in the presence of various concentrations of dasatinib (0.01-1μM) for 4 hours. Thereafter, Western blot analysis was performed using Abs directed against p-Lyn^Tyr507, Lyn, p-Btk^Tyr223, Btk, Hck, p-STAT5, STAT5, and KIT. To detect Hck and KIT activation, immunoprecipitation was performed using an anti–Hck-Ab or anti-KIT Ab, followed by immunoblotting with anti-phospho tyr-Ab 4G10. (B) HMC-1.2 cells were incubated in control medium (no drugs: 0) or in medium containing midostaurin and dasatinib (100-300nM each as indicated) either as single drugs or in combination for 48 hours. Thereafter, the number of apoptotic cells was counted by light microscopy. Results represent the mean ± SD of 3 independent experiments. *P < .05 compared with control (0). The bottom right panel shows combination index (CI) values obtained in these experiments using Calcusyn Software. As visible, the CI values are all below 1.0 indicating synergistic drug interactions.