Fig. 3.

MG132 induces caspase-8 oligomerization and aggregation. (A) Schematic representation of the full-length and truncated caspase-8 BiFC constructs. (B) bax^−/− bak^−/− BMK cells were transiently transfected with plasmids encoding C8 (C360S)-VN and C8 (C360S)-VC for 18 h, followed by treatment with MG132 for an additional 18 h. Representative fluorescence images were taken. (C) Cells untransfected, or transfected with the Venus pairs of C8 (C360S) were left untreated or were treated with MG132 for 18 h. The percentages of Venus green cells were analyzed by flow cytometry. The number of Venus-positive cells in MG132-treated samples was normalized against that of the untreated samples. The average of four experiments plus the SEM are shown. (D) Lysates from untreated or MG132-treated bax^−/− bak^−/− BMK cells were resolved by size exclusion chromatography on a Superdex 200 column. Eluate was collected in 0.5-ml fractions, concentrated by acetone precipitation, and probed for indicated proteins. The molecular masses of the protein standards used to indicate the approximate sizes of the fractions are indicated in kilodaltons.