Cellular Bcr-Abl tyrosine phosphorylation is reduced in the presence of AP23464 except in the case of T315I. Parental Ba/F3 cells or Ba/F3 cells expressing WT or mutant Bcr-Abl (4 × 106 cells/well) were incubated in escalating concentrations of AP23464 (0, 5, 10, 20, 40, 80, 160, or 320 nM) for 3 hours at 37° C. Clarified whole-cell lysates were subjected to SDS-PAGE followed by Western blot analysis. (A) Mouse monoclonal antiphosphotryrosine antibody 4G10 (Upstate Biotechnology) was used to determine levels of tyrosine-phosphorylated Bcr-Abl. Total Bcr-Abl expression levels were determined with mouse monoclonal anti-Abl antibody 8E9 (BD Biosciences Pharmingen) or Ab-2 (Oncogene Science). (B) Rabbit polyclonal antiphospho-Src family antibody (Cell Signaling Technology) was used to determine levels of tyrosine-phosphorylated Src family kinase members. As a loading control, total Lyn expression levels were determined with rabbit polyclonal anti-Lyn antibody sc-15 (Santa Cruz Biotechnology).
