Down-regulation of Bcr/Abl protein by WP1130 is not associated with changes in Hsp70 and is not suppressed by protease inhibition. (A) BV173 CML cells (wild-type Bcr/Abl) and BV173R cells (T315I Bcr/Abl mutant) (1 × 10⁶ cells/mL) were treated with 5 μM WP1130 for 1 hour or 40 μM of the proteasomal inhibitor MG-132 for 2 hours before treatment with WP1130 (5 μM) for 1 hour. Cells were harvested, and the expression of Bcr/Abl, pStat5, Stat5, pHck, and Hck was determined by Western blotting. Actin was used as a protein-loading control. (B) MM-1 multiple myeloma cells were treated with 5 μM WP1130 for 1 hour or pretreated with 40 μM MG-132 for 2 hours before treatment with WP1130 for 1 hour. Untreated and vehicle-treated (DMSO) cells served as controls. Cell lysates were immunoblotted for c-Myc. Actin was used as a protein-loading control. (C) K562 cells (1 × 10⁶) were treated with 5 μM WP1130 for 1 hour or pretreated with protease inhibitors (40 μM MG-132, 100 μM LLnL, 2500 μM NH₄Cl, 20 μM antipain, 40 μM zVAD) for 2 hours before incubation with WP1130 for 1 hour. Lysates were immunoblotted with anti-Abl to detect Bcr/Abl and c-Abl. pStat5 was also immunoblotted to determine the effect of inhibitors on Bcr/Abl signaling. (D) K562 cells (1 × 10⁶) were treated with 5 μM WP1130 or 5 μM geldanamycin (GA) for 1 hour or 24 hours before analyzing lysates for expression of Bcr/Abl and Hsp70 protein by Western blotting. Actin was used as a protein-loading control. (E) K562 cells (1 × 10⁶) were treated with 5 μM WP1130 for 1 hour or 2 hours, and the expression of Bcr/Abl and Hsp70 proteins in the treated and untreated cells was determined by Western blotting. Actin was used as a protein-loading control.