American Society of Hematology

Figure 4

$Figure 4. Prevention of Bax degradation by tBid at the mitochondrial level. (A) Determination of proteasome activity. (Ai) Proteasome activity was measured in both cytosol (Cyto) and mitochondrial (Mito) fractions in the absence of ubiquitin. *Significantly different proteasome activities (P < .0001) between DHL-4 and K562. (Aii) Inhibition of proteasome activity by bortezomib. Bortezomib (70 nM) was added to the Cyto (5 μg/μL protein) 1 hour before the reaction. Data shown were from 3 separate experiments. *Significant inhibition (P < .0001; t test). (B) Mito Bax degradation in K562 Cyto. K562 Mito were incubated with K562 Cyto in the presence of ubiquitin. Mito were mixed with Cyto according to a protein ratio of Mito/Cyto of 1:2 and incubated at 30°C for 3 hours. Bax degradation was monitored in the presence or absence of 10 nM tBid. Bax protein levels were detected by Western blotting for both Mito and Cyto. (C) K562 Mito were either incubated in the degradation buffer containing ubiquitin or in the K562 Cyto without ubiquitin for 3 hours. Bax protein levels were examined in the Mito fraction. (D) Mito Bax degradation in the DHL-4 Cyto. Mito were isolated from the K562 cells. Cyto was extracted from DHL-4 cells. In this assay, ubiquitin were omitted to test whether the DHL-4 Cyto has a higher Bax degradation activity. Mito were mixed with Cyto according to a protein ratio of Mito/Cyto of 1:2 and incubated at 30°C for up to 60 minutes in the presence or absence of 10 nM tBid. Bax protein levels were detected by Western blotting for both Mito and Cyto. (E) Bortezomib prevents Bax degradation. DHL-4 cells were treated with 100 nM bortezomib for 24 hours, and its Cyto was mixed with K562 Mito and incubated at 30°C for 5 hours. Mito Bax levels were then determined by Western blotting after separation from Cyto. Bax antibody (clone 2D2) was used for the Western blotting.$

Prevention of Bax degradation by tBid at the mitochondrial level. (A) Determination of proteasome activity. (Ai) Proteasome activity was measured in both cytosol (Cyto) and mitochondrial (Mito) fractions in the absence of ubiquitin. *Significantly different proteasome activities (P < .0001) between DHL-4 and K562. (Aii) Inhibition of proteasome activity by bortezomib. Bortezomib (70 nM) was added to the Cyto (5 μg/μL protein) 1 hour before the reaction. Data shown were from 3 separate experiments. *Significant inhibition (P < .0001; t test). (B) Mito Bax degradation in K562 Cyto. K562 Mito were incubated with K562 Cyto in the presence of ubiquitin. Mito were mixed with Cyto according to a protein ratio of Mito/Cyto of 1:2 and incubated at 30°C for 3 hours. Bax degradation was monitored in the presence or absence of 10 nM tBid. Bax protein levels were detected by Western blotting for both Mito and Cyto. (C) K562 Mito were either incubated in the degradation buffer containing ubiquitin or in the K562 Cyto without ubiquitin for 3 hours. Bax protein levels were examined in the Mito fraction. (D) Mito Bax degradation in the DHL-4 Cyto. Mito were isolated from the K562 cells. Cyto was extracted from DHL-4 cells. In this assay, ubiquitin were omitted to test whether the DHL-4 Cyto has a higher Bax degradation activity. Mito were mixed with Cyto according to a protein ratio of Mito/Cyto of 1:2 and incubated at 30°C for up to 60 minutes in the presence or absence of 10 nM tBid. Bax protein levels were detected by Western blotting for both Mito and Cyto. (E) Bortezomib prevents Bax degradation. DHL-4 cells were treated with 100 nM bortezomib for 24 hours, and its Cyto was mixed with K562 Mito and incubated at 30°C for 5 hours. Mito Bax levels were then determined by Western blotting after separation from Cyto. Bax antibody (clone 2D2) was used for the Western blotting.

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