Effect of KIT D816V on Lyn and Btk activation in HMC-1 cells and Ba/F3 cells. (A) HMC-1.1 and HMC-1.2 cells were kept in control medium or in the presence of 1μM midostaurin for 4 hours. Thereafter, Western blot (WB) analysis was performed using Abs directed against p-Lyn^Tyr507, p-Src^Tyr416, Lyn, p-Btk^Tyr223, Btk, and KIT. Moreover, the phosphorylation of KIT was analyzed by immunoprecipitation using an anti-KIT Ab followed by immunoblotting using an anti-phosphotyrosine Ab. The β-actin control is also shown. To confirm the specificity of this WB result, we also performed immunoprecipitation experiments using anti-Lyn Ab followed by immunoblotting with anti-pSrc^Tyr416 and anti-pLyn^Tyr507 as well as anti-phospho-Ab 4G10. Again, midostaurin failed to suppress expression of p-Lyn (not shown). (B) Ba/F3 cells with doxycycline-inducible expression of wt KIT (Ton.Kit.wt) or KIT D816V (Ton.Kit.D816V) were kept in control medium or in the presence of doxycycline (doxy, 1 μg/mL) for 24 hours. In case of Ton.Kit.wt, SCF (100 ng/mL) was added to induce KIT phosporylation for 15 minutes. Thereafter, cells were harvested and subjected to Western blot analysis using an anti-phosphotyrosine Ab for detection of activated KIT and Abs directed against p-Lyn^Tyr507, p-Src^Tyr416, Lyn, p-Btk^Tyr223, Btk, and phosphorylated STAT5 (p-STAT5) as well as total STAT5. Ba/F3 cells stably expressing BCR/ABL T315I (Ba/F3p210^T315I) served as a positive control. The dual bands observed with Abs against Lyn and STAT5 in Ton.Kit.D816V cells may be explained by alternative splicing.