Dasatinib binds to and blocks phosphorylation of Btk in human basophils. (A) Anti-ABL and anti-BTK immunoblots of KU812 pulldowns with dasatinib and ampicillin (negative control). The samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 7% gel. Regions corresponding to BCR-ABL (∼ 210 kDa), c-ABL (∼ 145 kDa), and BTK (∼ 75 kDa) are displayed. TCL indicates total cell lysate (120 μg TCL protein loaded). (B) Immunoblot of lysates derived from KU812 cells after cells had been exposed to control medium or dasatinib (1 μM) at 37°C for 4 hours. Antibodies against phosphorylated Btk (p-Btk), p-Hck, total Btk, and total Lyn were applied as indicated. For detection of p-Lyn, immunoprecipitation was performed using total Lyn antibody and antiphosphotyrosine 4G10 antibody. (C) Basophils obtained from 2 leukemia patients with basophilia (AML, left panel; CML, right panel) were preincubated with control medium or dasatinib (3 μM) at 37°C for 15 minutes, and then were exposed to an anti-IgE antibody (1 μg/mL) at 37°C for 10 minutes. After incubation, cells were recovered and subjected to Western blotting using antibodies against p-Lyn and total Lyn protein. Western blotting was performed as described in the text.