WP1130 stimulates Bcr-Abl ubiquitination and trafficking in CML cells. (A) WDT-2 cells were treated with vehicle alone (control) or 5μM WP1130 (WP) for 30 minutes at 37°C before an equal volume of cell lysates were resolved into total (T), detergent-soluble (S), or detergent-insoluble (I) fractions and probed for Bcr-Abl (left) or ubiquitin (center). Equal protein (400 μg) detergent-soluble cell lysate from WP1130-treated or control cells was treated with 1% SDS at 60°C, diluted to 0.1% SDS, and subjected to immunoprecipitation with anti-Abl (K12). The immunoprecipitate was washed, resolved on gels, and immunoblotted for Abl (left) or ubiquitin (right). (B) K562 or BV-173 cells were treated as described in panel A, and equal amounts of detergent-soluble cell lysates were subjected to Abl immunoprecipitation and blotting for Abl and ubiquitin (as described in panel A right). An aliquot of the same lysate (200 μg) from control and treated cells was also subjected to affinity enrichment for K48-linked (ataxin) or K63-linked (Rap80) ubiquitin polymers. Bound protein was eluted and subjected to Abl immunoblotting. (C) eGFP-Bcr-Abl–transformed BaF3 cells were treated with vehicle alone (top panel) or with 5μM WP1130 for 4 hours (bottom panel) before cells were fixed, cytospun onto slides, and permeabilized. After blocking reactive sites, slides were incubated with anti-ubiquitin antibody (1:100), washed, and antigen detected with Alexa Fluor antibodies. The slides were washed again and stained for nucleus detection with Hoechst 33342. Images were acquired using an Olympus confocal microscope FV-500 and merged with Photoshop Version 6.0 software. The arrows depict Bcr-Abl/ubiquitin colocalization, and their juxtanuclear localization resembles aggresomes.