Correlation between direct binding of Bag1 and BCR-ABL degradation. (A) GST-(−), -CHIP, -Hsc70, -Hsp90, -cdc37, -Bag1M, -Bag1S, or -p23 were incubated with in vitro–transcribed/translated BCR-ABL with (+) or without (−) GA (10μM) or imatinib (10μM). The proteins associated with those GST-fusion proteins were analyzed by immunoblotting with the indicated antibodies. Vertical lines are inserted to indicate a repositioned gel lane. Because of a large number of samples, Western blotting was performed using 2 separate gels with completely identical experimental conditions. (B-D) GST-Bag1 was incubated with BCR-ABL, BCR, ABL (B), Flag-tagged ABL kinase domain fragments (wt and KD; C), and BCR 1-413 (D) that were in vitro–transcribed/translated in the presence (+) or absence (−) of GA (10μM) in RRLs. GST-Bag1–bound proteins were analyzed by immunoblotting with the indicated antibodies. (E) COS7 cells were transiently transfected with Flag-tagged BCR-ABL wt, KD, or Δkina together with Xpress (Xp)–tagged Bag1. Twenty-four hours after transfection, anti-Flag immunoprecipitates were immunoblotted with antibodies against Xpress and Flag. (F) K562 cells were treated with GA (1μM) for 4 hours. Anti-Bag1 or mouse control immunoglobulin G (IgG) immunoprecipitates were immunoblotted with antibodies against ABL and Bag1. (G) His-tagged ABL kinase domain fragments or His-tagged Hsc70 proteins coated on microtiter plates were incubated with various concentrations of His-tagged Bag1 and bound Bag1 was detected by anti-Bag1 antibody.