Fig. 3.
TNF- induced Bcl-2 protein translocation from light membrane to the mitochondrial outer membrane. CEM and CEM/VLB100 cells were treated with 250 U/mL TNF- for 3 hours. (A) Freshly isolated mitochondria were blocked with BSA, labeled with anti–Bcl-2 antibody, FITC-conjugated anti-mouse antibody, and analyzed with flow cytometry. Control mouse IgG served as a negative control. (B) Light membrane was dissolved with lysis buffer, and 50 μg protein was used for Western blotting. Protein concentration of light membrane was confirmed by Bradford assay and by measuring optical density 280 nm at 260 nm (RNA concentration). Ratio of Bcl-2, control:TNF for CEM = 61:39 (1.6:1) for CEB/VLB100=75:25 (3:1).
