Protein isolation and Western blotting
For protein isolation portions of liver were sonicated in homogenization buffer containing
1 mmol/L sodium bicarbonate, 0.5 mmol/L calcium chloride and protease and
phosphatase inhibitors as previously described.1 Western blotting was performed by
denaturing 50 g of protein at 100°C for 5 min in Laemmli sample buffer containing 62.5
mmol/L Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue and 5% mercaptoethanol.
Proteins were resolved by SDS-PAGE and transferred to a
nitrocellulose membrane (Schleicher & Schuell, Keene, NH) in transfer buffer containing
25 mmol/L Tris, pH 8.3, 192 mM glycine, 0.01% SDS, and 15% methanol using a BioRad (Hercules, CA) Trans-blot SD semidry transfer cell to which 150 mA were applied
for 90 min. Membranes were blocked in 5% non-fat dry milk, 20 mM Tris, pH 7.5, 500
mM sodium chloride, and 0.5% Tween 20 (TBS-T) for 1 h. Rabbit anti-phospho-c-Jun
(Cell Signaling, Beverly, MA), rabbit anti-c-Jun, mouse anti-phospho-JNK and rabbit
anti-JNK (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used as primary
antibodies at 1:1,000 to 1:2,000 dilutions in 5% bovine serum albumin for 18 h at 4°C.
Membranes were exposed to goat anti-rabbit and anti-mouse secondary antibodies
conjugated with horseradish peroxidase (KPL, Gaithersburg, MD) at a dilution of
1:10,000 in 5% non-fat milk TBS-T for 1 h at room temperature. Signals were detected
with a chemiluminescence detection system (Western Lightning Chemiluminescence
Plus, PerkinElmer, Boston, MA) and exposure to x-ray film.
Electrophoretic mobility shift assay
Nuclear proteins were isolated as previously described.2 Electrophoretic mobility shift
assays (EMSA) were performed with an oligonucleotide for the activator protein (AP)-1
consensus sequence (Santa Cruz Biotechnology). The DNA binding reaction was
performed at room temperature for 20 min in a 20 µL reaction mixture consisting of 5 µg
of nuclear extract, 50 µg/mL of polydeoxyinosinic-deoxycytidylic acid, 10 mM Tris, pH
7.5, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mg/mL bovine serum albumin,
10% glycerol, and the [32P]-end-labeled oligonucleotide. After incubation, the samples
were resolved on a 4% polyacrylamide gel, dried and subjected to autoradiography.
JNK activity assay
JNK activation was measured in cell lysates using a JNK in vitro kinase activity assay
according to the manufacturer’s instructions (Cell Signaling). A c-Jun fusion protein was
used to precipitate JNK from cell lysates containing 200 g of cellular protein. After
washing, kinase reactions were performed in the presence of 10 mmol/L ATP with a cJun fusion protein as substrate. Samples were resolved in 10% SDS polyacrylamide gels
and immunoblotting performed as previously described. The relative amounts of the
phosphorylated c-Jun fusion protein were detected with a phospho-c-Jun antibody. As a
control for the loading of equivalent amounts of protein among samples, levels of total cJun were also determined.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from whole liver by cesium chloride gradient centrifugation, as
previously described.3 The expression levels of intracellular adhesion molecule-1
(ICAM-1), monocyte chemoattractant protein-1 (MCP-1) and -actin were determined by
RT-PCR using the QIAGEN (Valencia, CA) OneStep RT-PCR kit according to the
manufacturer’s instructions. Briefly 50 L reactions were carried out with a mixture of
400 mol/Lof each dNTP, 0.6mol/Lof the forward and reverse primers, 2 L of the
QIAGEN OneStep enzyme mix and 1g of total RNA. The sequences of the primers
used were: ICAM-1 (forward) 5′-GGA GCA AGA CTG TGA ACA CG-3′, (reverse) 5’GAG AAC CAC TGC TAG TCC AC-3′; MCP-1 (forward) 5′-ACT GAA GCC AGC
TCT CTC TTC CTC-3′, (reverse) 5′-TTC CTT CTT GGG GTC AGC ACA GAC-3′; actin (forward) 5′-ATG GAT GAC GAT ATC GCT G-3′, (reverse) 5′-ATG AGG TAG
TCT GTC AGG T-3′. The PCR products were subjected to 1.5% agarose gel
electrophoresis with ethidium bromide, visualized under UV light and scanned images
quantitated by densitometry. Transcripts of constitutively expressed -actin were used to
normalize the levels of ICAM-1 and MCP-1 mRNA in each sample.
References:
1. Schattenberg JM, Wang Y, Rigoli RM, Koop DR, Czaja MJ. CYP2E1 overexpression
alters hepatocyte death from menadione and fatty acids by activation of ERK1/2
signaling. Hepatology 2004; 39:444-455.
2. Xu Y, Bradham C, Brenner DA, Czaja MJ. Hydrogen peroxide-induced liver cell
necrosis is dependent on AP-1 activation. Am J Physiol 1997; 273:G795-G803.
3. Liu H, Lo CR, Jones BE, Pradhan Z, Srinivasan A, Valentino KL et al. Inhibition of cMyc expression sensitizes hepatocytes to tumor necrosis factor-induced apoptosis
and necrosis. J Biol Chem 2000; 275:40155-40162.