Horseradish peroxidase coupled anti rabbit igg

Western blotting was performed, as previously described (Bajt et al., 2000 (link)). The primary antibodies were rabbit anti-JNK and anti-phospho-JNK antibodies (Cell Signaling Technology, Danvers, MA), rabbit anti-Bax polyclonal antibody (Cell Signaling Technology, Danvers, MA), rabbit anti-AIF antibody (Cell Signaling Technology, Danvers, MA), rabbit anti-EndoG (ProSci, Poway, CA) and rabbit anti-nitrotyrosine (Upstate, Lake Placid, NY). A horseradish peroxidase-coupled anti-rabbit IgG (Santa Cruz) was used as secondary antibody.

Mitochondria and cytosolic fractions were isolated as described (Cover et al., 2005 (link)). Briefly, the liver was homogenized in ice cold isolation buffer (pH7.4) containing 220 mM mannitol, 70 mM sucrose, 2.5 mM HEPES, 10 mM EDTA, 1 mM EGTA, and 0.1% bovine serum albumin. Mitochondria were isolated by differential centrifugation (20,000 xg) and washed with 2 ml of isolation buffer. The 20,000 xg supernatant was used to evaluate the release of mitochondrial factors such as apoptosis-inducing factor (AIF) by western blotting. Western blotting was carried out as described in detail (Bajt et al., 2000 (link)) using the following antibodies: a rabbit anti-AIF antibody (Abcam, Cambridge, MA), a rabbit anti-Cyp2E1 polyclonal antibody (Abcam, Cambridge, MA), a mouse anti-Bcl-xl monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit anti-LC-3 polyclonal antibody (MBL, Naka-ku Nagoya, Japan), a mouse anti-GADD 153/CHOP monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a rat anti-Grp 78 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse monoclonal anti-metallothionein antibody (DAKO Corp., Carpinteria, CA). A horseradish peroxidase-coupled anti-rabbit IgG (Santa Cruz) was used as secondary antibody. Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech. Inc., Piscataway, NJ).