Total jnk

Nuclear extracts, cytosol extracts, or total proteins of liver tissue were prepared as a published method with modification [19 (link)]. The protein concentration was measured by the BCA assay. Aliquots of protein (30 μg) were denatured at 95°C for 5 min before electrophoresis on 10% SDS-polyacrylamide gel. After transfer to a polyvinylidene difluoride (PVDF) membrane (Millipore), the blot was blocked with 5% nonfat milk solution for 1 h at room temperature and then incubated with a 1 : 1,000 dilution of primary antibodies selective against either NF-κB p65, total JNK, p-JNK, total ERK, p-ERK, total Akt, p-Akt, total p38, p-p38, histone, or β-actin (Santa Cruz Biotechnology) in Tris-buffered saline Tween-20 (TBST) at 4°C overnight, followed by 1 h at room temperature. The membrane was washed 3 times for 5 min each with TBST solution. The membranes were incubated with 1 : 10,000 dilution of horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. The transferred proteins were visualized with an enhanced chemiluminescence (ECL) detection system and the band intensities were determined using a Gel Doc/ChemiDoc imager (Azure). The protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA).

Equal amounts of lysates were resolved by sodium dodecyl-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The blots were visualized using an enhanced chemiluminescence system (Amersham Biosciences Inc., Piscataway, NJ, USA) followed by exposure to X-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan), as previously described [25 (link)]. Primary antibodies against iNOS, total p38, phosphorylated JNK, phosphorylated ERK1/2, total JNK, total ERK1/2, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phosphorylated IκB-α, phosphorylated IKK-α/β, and phosphorylated p38 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).