Figure 4

The status of calpain in TK VI-treated cells. (A) Protein samples derived from HepG2 cells treated with 20 μM TK VI for 8 h were analyzed by immunoblotting. The 80-kDa pro-form of μ-calpain appeared as an abundant band, and the cleaved 78-kDa form of the enzyme was visible in some samples just below the pro-form. (B) HepG2 cells were pretreated with BAPTA and SJA6017, followed by 20 μM TK VI for 8 h. Then activation of Bak was detected with activated N-terminal anti-Bak antibody and mitochondria were visualized with red fluorescent protein (Mito-red) by imunofluorescence microscopy. (C) The cells were incubated with TK VI as described in (B). Mitochondria were visualized with red fluorescent protein (Mito-red). The redistribution of Bax was marked with FITC-conjugated anti-mouse and assessed by confocal immunofluorescence. (D) The cells were incubated with TK VI as described in (B). Equal amounts of mitochondrial proteins was isolated from HepG2 cells and then subjected to immunoblot analysis. Cox IV was used as a marker for mitochondrial fraction.