FIG. 4.
Effects of Mcl-1 and Bcl-XL overexpression on the proapoptotic property of the core protein. (A) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3 in Huh7 cells that were transfected with Bcl-2, Bcl-XL, Bcl-w, Mcl-1, or vector only. All experiments were performed in triplicate, and the average values with standard deviations are plotted. (B) Western blot analysis also was performed to determine the cleavage of endogenous PARP (top) and expression levels of the myc-tagged prosurvival members of the Bcl-2 family (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom). (C) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3 in Huh7 cells that were singly transfected with vector, the wild-type core protein, Mcl-1, or Bcl-XL, or that were cotransfected with wild-type core protein and Mcl-1 or Bcl-XL. The amounts of flag-core and myc-Mcl-1 or myc-Bcl-XL DNAs used in each of the transfections are indicated in micrograms. In each transfection, the total amount of DNA was normalized to 3 μg with the addition of empty vector if necessary. All experiments were performed in triplicate, and the average values with standard deviations are plotted. (D) Western blot analysis also was performed to determine the cleavage of endogenous PARP (top) and expression levels of myc-tagged Mcl-1 and Bcl-XL and flag-tagged core protein (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom).