Horseradish peroxidase coupled donkey anti rabbit igg

Mitochondria and cytosolic fractions were isolated using differential centrifugation as described (Xie et al., 2013 (link)). Western blots were performed as described in detail (Bajt et al., 2000 (link); Du et al., 2013 (link)), using the following antibodies: a rabbit anti-JNK antibody (Cell signaling Technology, Danvers, MA), a rabbit anti-phospho-JNK antibody #4668, which recognizes Thr183/Tyr185 phosphorylated JNK1/2 protein (Cell Signaling Technology, Danvers, MA). A horseradish peroxidase-coupled donkey anti-rabbit IgG (Santa Cruz) was used as secondary antibody. Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech. Inc, Piscataway, NJ).

Expression of selected genes quantified by real-time PCR (RT-PCR) analysis was performed as described previously (Bajt et al., 2008 (link)). Briefly, total RNA was extracted from liver tissue using TRI reagent (Sigma, St Louis, MO), reversed transcribed into cDNA using random primers and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) at 0.1μg/μl. The cDNA was diluted 1/10 and 5μl was used as a template in each PCR reaction. SYBR green PCR Master Mix (Applied Biosystems) was applied as the detector. mRNA of glutamate cysteine ligase catalytic subunit (gclc) was evaluated by normalizing to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and then expressed as a fold increase relative to control (arbitrarily set as 1.0). Western blotting was performed as described in detail (Bajt et al., 2000 (link)). The antibodies used for JNK were: rabbit anti-JNK and anti-phospho-JNK antibodies (Cell Signaling Technology, Danvers, MA), horseradish peroxidase-coupled donkey anti-rabbit IgG (Santa Cruz).