Geldanamycin and 17-AAG induce degradation of wild-type and imatinib mesylate–resistant, mutant BCR-ABL proteins and inhibit BCR-ABL signaling.

(A) Ba/F3 cells expressing wild-type, T315I, or E255K BCR-ABL were incubated in the presence of increasing concentrations of geldanamycin (GA) for 24 hours. Immunoblotting of cell lysates was performed with anti-ABL (Ab3, Oncogene, San Diego, CA) (upper panels), anti–RAF-1 (Santa Cruz Biotechnology, Santa Cruz, CA) (middle panels), and antiactin (ac-15, Sigma) as a control for protein loading (lower panels). (B) Ba/F3 cells expressing wild-type, T315I, or E255K BCR-ABL were incubated in the presence of increasing concentrations of 17-AAG for 24 hours. Immunoblotting of these lysates was performed with anti-ABL (upper panels) and antiactin as a control for protein loading (lower panels). (C) Immunoblotting of the same lysates from (B) was performed with anti-CRKL (Santa Cruz). CRKL, when tyrosine-phosphorylated, migrates more slowly on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) resulting in an upper band representing phosphorylated CRKL (P-CRKL) and a lower band representing nonphosphorylated CRKL. (D) Densitometric analysis of CRKL immunoblot shown in (C) using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Quantified CRKL phosphorylation is expressed as percentage of phosphorylated CRKL over total CRKL protein (% P-CRKL). (E) Densitometric analysis of CRKL immunoblot using lysates from the same Ba/F3 cell lines incubated in the presence of increasing concentrations of imatinib mesylate for 24 hours.