Measurement of the mitochondrial apoptosis pathway

The mitochondrial apoptosis pathway was assessed by measuring caspase activity, cytochrome c release from the mitochondria, and bcl-2 levels in the liver obtained from young (6–7 months old) Tg(TXN)^+/0 and WT mice. The caspase activities, cytochrome c release, and bcl-2 levels were determined by Western blot assay. The caspase-3 activity in liver tissue were visualized by the cleavage forms of caspase-3 (p20) as an indicator of caspase activity using anti-caspase-3 antibody (Cell Signaling Technology, Inc., Danvers, MA). The intensities of cleavage bands corresponding to p20 for caspase-3 were quantified by densitometry using ImageQuant v5.0, and β-actin was used as a loading control. The cytochrome c release from mitochondria was measured as levels of cytochrome c in the cytosolic fractions from liver. Equal amounts of protein were separated on a 4–20% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and subjected to Western blotting with an anti-cytochrome c antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The bcl-2 levels were also measured by Western blot using anti-bcl-2 antibody (Cell Signaling Technology, Inc., Danvers, MA). Data were normalized to β-actin loading controls. The data were expressed as relative units respective to β-actin.