Figure 6
Figure 6. Bag1 stimulates CHIP-induced BCR-ABL degradation and combination of Bag1 overexpression and Hsc70 knockdown promotes CHIP-induced suppression of BCR-ABL–dependent cell growth. (A) Flag-tagged BCR-ABL proteins were in vitro transcribed/translated with (+) or without (−) GA and then incubated with 20S proteasome with (+) or without (−) His-tagged Bag1. Numbers indicate the intensities of the bands of 20S proteasome. (B) Flag-tagged BCR-ABL proteins were in vitro–transcribed/translated with GA and then incubated with 20S proteasome with His-tagged Bag1 (wt) or His-tagged ubiquitin like domain-deletion mutant of Bag1 (ΔUb). Anti-Flag immunoprecipitates and whole reticulocyte lysates (WRL) were subjected to immunoblotting with the indicated antibodies (A-B). (C) Glutathione sepharose-bound GST-tagged Bag1 proteins were incubated with His-tagged CHIP, E1, E2 (UbcH5b or UbcH5c), and biotin-labeled ubiquitin. Ubiquitinated proteins were detected by streptavidin-HRP. An arrow indicates ubiquitinated Bag1. (D) COS7 cells were transiently transfected with the BCR-ABL together with or without Xpress-tagged Bag1M, Bag1S, or control vector, and the tet system of CHIP or control vector. (E) BCR-ABL–expressing Ba/F3 cells with the tet system for CHIP were transiently transfected with Xpress-tagged Bag1S or Bag1M together with or without Hsc70 siRNA. The cells were incubated with or without tet for 24 hours (D) or 48 hours (E) and were analyzed by immunoblotting with the indicated antibodies (D-E). (F-G) BCR-ABL–expressing Ba/F3 cells with the tet system for CHIP were transiently transfected with Xpress-tagged Bag1S or Bag1M together with or without Hsc70 siRNA. After 2 days, the viable cell numbers were analyzed based on volume and side-scattering gating (F) and then GFP-positive (CHIP-expressing) cell numbers were analyzed by fluorescent intensity (G) with flow cytometry. (H) K562 cells were transiently transfected with the tet system for CHIP together with or without Xpress-tagger Bag1 and Hsc70 siRNA. After 2 days, the viable cell numbers were analyzed based on volume and side-scattering gating with flow cytometry. The values are mean ± SEs determined by 4 independent experiments. *P < .05, **P < .01, ***P < .001.

Bag1 stimulates CHIP-induced BCR-ABL degradation and combination of Bag1 overexpression and Hsc70 knockdown promotes CHIP-induced suppression of BCR-ABL–dependent cell growth. (A) Flag-tagged BCR-ABL proteins were in vitro transcribed/translated with (+) or without (−) GA and then incubated with 20S proteasome with (+) or without (−) His-tagged Bag1. Numbers indicate the intensities of the bands of 20S proteasome. (B) Flag-tagged BCR-ABL proteins were in vitro–transcribed/translated with GA and then incubated with 20S proteasome with His-tagged Bag1 (wt) or His-tagged ubiquitin like domain-deletion mutant of Bag1 (ΔUb). Anti-Flag immunoprecipitates and whole reticulocyte lysates (WRL) were subjected to immunoblotting with the indicated antibodies (A-B). (C) Glutathione sepharose-bound GST-tagged Bag1 proteins were incubated with His-tagged CHIP, E1, E2 (UbcH5b or UbcH5c), and biotin-labeled ubiquitin. Ubiquitinated proteins were detected by streptavidin-HRP. An arrow indicates ubiquitinated Bag1. (D) COS7 cells were transiently transfected with the BCR-ABL together with or without Xpress-tagged Bag1M, Bag1S, or control vector, and the tet system of CHIP or control vector. (E) BCR-ABL–expressing Ba/F3 cells with the tet system for CHIP were transiently transfected with Xpress-tagged Bag1S or Bag1M together with or without Hsc70 siRNA. The cells were incubated with or without tet for 24 hours (D) or 48 hours (E) and were analyzed by immunoblotting with the indicated antibodies (D-E). (F-G) BCR-ABL–expressing Ba/F3 cells with the tet system for CHIP were transiently transfected with Xpress-tagged Bag1S or Bag1M together with or without Hsc70 siRNA. After 2 days, the viable cell numbers were analyzed based on volume and side-scattering gating (F) and then GFP-positive (CHIP-expressing) cell numbers were analyzed by fluorescent intensity (G) with flow cytometry. (H) K562 cells were transiently transfected with the tet system for CHIP together with or without Xpress-tagger Bag1 and Hsc70 siRNA. After 2 days, the viable cell numbers were analyzed based on volume and side-scattering gating with flow cytometry. The values are mean ± SEs determined by 4 independent experiments. *P < .05, **P < .01, ***P < .001.

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