Bag1 mediates Hsp90 inhibitor-induced BCR-ABL degradation. (A) GST-Bag1 was incubated with SH2 kinase domain fragments (wt and T315I) 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. (B) COS7 cells were transiently transfected with Flag-tagged kina-wt and -T315I. Twenty-four hours after transfection, cells were treated with imatinib and GA for 8 hours. (C) Ba/F3 cells expressing p185 BCR-ABL-wt were preincubated with imatinib (10μM) for 30 minutes and with GA (3μM), Rad (3μM), and 17-AAG (3μM) for additional 8 hours. The culture was performed with IL-3 to guarantee the cell survival after BCR-ABL inhibition. The bottom graph shows quantification of relative amounts of BCR-ABL proteins. (D) Twenty-four hours after transfection of anti-Bag1 siRNA to K562 cells, GA was added for 8 hours. Numbers indicate the intensities of the bands of BCR-ABL. The cell lysates were analyzed by immunoblotting with the indicated antibodies (B-D).