Effect of bortezomib on proteasome and BCR-ABL activity. (A) CML CD34⁺ samples (n = 2) labeled X and Y were treated for 24 hours with bortezomib 10 or 20nM (10,20) and compared with an untreated control (ND). Accumulation of polyubiquitinated proteins of various molecular weights was seen. Protein loading was assessed using antiactin antibody. The presence of significant BCR-ABL activity represented by the pCrkl band can be seen in all samples. (B) A representative CML CD34⁺ sample treated with bortezomib 10 or 20nM (10, 20) or dasatinib 150nM (DAS) for 24 to 48 hours and compared with untreated control (ND). The presence of the pCrkl band demonstrates the significant residual BCR-ABL kinase activity in PI-treated samples compared with the abrogated activity in the TKI-treated sample. (C) Densitometry of pCrkl (■) and ubiquitin (□) relative to protein loading control for 3 samples is shown after bortezomib 10nM (B10), dasatinib 150nM (D150), and combination treatment (mean ± SEM).