Bax protein instability. (A) Expression of Bcl-2, Bcl-XL, Bak, and Bax proteins. A total of 25 μg proteins/each lane was loaded to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The anti–Bcl-2 (100) and anti–Bcl-XL antibodies were used at 1:200 dilution; anti-Bax (2D2) antibody was used at 1:1000 dilution, anti-Bak antibody was used at 1:200 dilution, and anti–β-actin antibody was used at 1:10 000 dilution. (B) RT-PCR detection of Bax mRNA levels. (C) The lifespan of Bax protein. HRC, CRL, or DoHH2 cell lines were preincubated with or without 50 nM bortezomib (Borte) for 2 hours and then treated with 50 μg/mL CHX for 3 hours. Proteins were extracted hourly. Bax levels were evaluated by Western blotting using Bax 2D2 antibody at 1:1000 dilution. β-Actin antibody was used at 1:10 000 dilution. Numbers under each pair of blots are the ratio of Bax/β-actin.