AP23464 inhibits tyrosine phosphorylation of Bcr-Abl and downstream targets STAT-5 and CrkL. K562 cells (1 × 10⁶) were incubated for 4 hours in 20 mL media in the presence of AP23464 (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, or 10 μM). Total cell lysates were prepared and separated by SDS-PAGE on 10% gels (50 μg protein/lane). Western blot analysis for phospho–Bcr-Abl, phospho-STAT5, phospho-CrkL, and eIF4E (protein loading control) was performed by using a Pathscan Bcr/Abl Multiplex Western Detection Kit (Cell Signaling Technology). Immunoblotting with nonphosphospecific antibodies confirmed that total levels of Bcr-Abl, STAT5, and CrkL were unchanged (data not shown).