Rabbit anti jnk antibody

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).

Mitochondria and cytosolic fractions were isolated as described (Cover et al., 2005 (link)). Briefly, aliquots of liver tissue or one of the kidneys were homogenized in ice cold isolation buffer (pH 7.4) 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×g) and washed with 2 ml of isolation buffer. The 20,000×g supernatant was used to measure JNK activation in the cytosol by western blotting in the liver and kidney as described (Akakpo et al., 2018 (link)), with the following Cell Signaling primary antibodies: rabbit anti-JNK antibody (Cat. # 9252) or rabbit anti-phospho-JNK antibody (Cat. # 4668). Liver and kidney samples were also used for western blotting with antibodies to NGAL (Santa Cruz, Cat. #sc-515876), cytochrome P450 2E1 (Abcam rabbit polyclonal antibody, cat. # ab28146) and ß-actin (Cell Signaling, Cat. # 4967). Anti-rabbit or anti-mouse IgG horseradish peroxidase coupled secondary antibodies (SantaCruz Biotechnology) (1:5000 dilution), coupled with the ECL kit from Amersham (Piscataway, NJ) were then used for detection of protein bands by chemiluminescence on the Licor Odyssey imaging system (LICOR Biosciences).