Hsc70 attenuates Bag1 binding BCR-ABL and inhibits CHIP-induced BCR-ABL degradation. (A) GST-Bag1 and an increasing amount of His-tagged Hsc70 were incubated with in vitro–transcribed/translated BCR-ABL in the presence (+) or absence (−) of His-tagged CHIP. The bottom graph shows quantification of relative amounts of BCR-ABL proteins. (B) BCR-ABL, a Flag-tagged ABL kinase domain fragment, or Flag-tagged BCR 1-413 was in vitro transcribed/translated in RRLs with GA and incubated with GST-Bag1 with (+) or without (−) His-tagged Hsc70. Bag1 and Hsc70 panels represent the 3 experiments with BCR-ABL, kina, and BCR 1-413 for each. (C) GST-Bag1 was incubated with in vitro–transcribed/translated BCR-ABL with (+) or without (−) GA (10μM) in RRLs immunodepleted of Hsc70. The proteins bound to GST-Bag1 were analyzed by immunoblotting with the indicated antibodies (A-C). (D) COS7 cells were transiently transfected with Flag-tagged BCR-ABL and Flag-tagged Hsp90, Hsc70, or control vector together with the tet system of CHIP, c-Cbl, or control vector. A vertical line indicates where lanes of the identical experiment were rearranged for consistency. (E) BCR-ABL–expressing Ba/F3 cells with the tet system for CHIP were transiently transfected with Flag-tagged Hsp90, Hsc70, Hsc54, or control vector. The cells were incubated with or without tet for 24 hours and were analyzed by immunoblotting with antibodies as indicated (D-E). The graph shows the ratios of BCR-ABL protein amount without tet (−) relative to that with tet (+) in E that were normalized against those of the control vector. The values are mean ± SEs. *P < .05.