To determine cell production of RANKL and OPG, hepatocytes or Kupffer cells were distributed onto 24-well flat-bottomed plates (Trasadingen, Switzerland) at a concentration of 2.0 × 105 cells/500 μL/well and incubated overnight to allow cell adherence. Cells were treated with 2, 10, or 50 ng/mL TNF-α for 8
or 24 hours. Culture media was collected and analyzed by way of ELISA kit for RANKL or OPG (R&D find protocol Systems). To evaluate NF-κB activation, primary hepatocytes or AML-12 (American Type Culture Collection [ATCC], Manassas, VA) cells were distributed onto a 100-mm dish at a concentration of 6 × 106 cells/10mL/dish for electrophoretic mobility shift assay (EMSA). Cells were treated with 10 ng/mL recombinant RANKL for 0.5, 1, 2, or
3 hours and harvested for nuclear extraction. Hepatocyte cytotoxicity was determined by lactate dehydrogenase (LDH) assay according to the manufacturer’s instructions (Roche, Mannheim, Germany). Primary hepatocytes were distributed onto 96-well flat-bottomed plates (Trasadingen) at a concentration of 1.5 × 104 cells/200 μL/well and incubated overnight to allow cell adherence. Cells were treated with 10 ng/mL recombinant RANKL for 24 hours. After removal of culture medium, cells were incubated with 50 ng/mL TNF-α and 200 mM hydrogen peroxide (H2O2) for 24 hours. Liver samples were homogenized in lysis buffer (10 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, 0.5 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL aprotonin, 10 μg/mL soybean trypsin inhibitor, 1 μg/mL pepstatin). Samples were then sonicated and incubated this website for 30 minutes on ice. Cellular debris was removed Alectinib by centrifugation at 10,000 rpm. Protein concentrations of each sample were determined. Samples containing equal amounts of protein in equal volumes of sample buffer were separated in a denaturing 10% polyacrylamide gel and transferred to a 0.1 μm pore nitrocellulose membrane. Nonspecific binding sites were blocked with Tris-buffered saline (TBS; 40 mM Tris, pH 7.6, 300 mM NaCl) containing 5% non-fat dry milk for 1 hour at room temperature. Membranes
were then incubated with antibodies to RANKL (R&D Systems) or Bcl-2 (Abcam, Cambridge, MA) in TBS with 0.1% Tween 20 (TBST). Membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase. Immunoreactive proteins were detected by enhanced chemiluminescence. Nuclear extracts of liver tissue were prepared by the method of Deryckere and Gannon23 and analyzed by EMSA. Briefly, double-stranded consensus oligonucleotides to NF-κB (Promega, Madison, WI) were end-labeled with g[32P] ATP (3,000 Ci/mmol at 10 mCi/mL; Perkin Elmer, Waltham, MA). Binding reactions (total volume 15 μL) containing equal amounts of nuclear protein extract (20 μg) and 35 fmols (≈50,000 cpm, Cherenkov counting) of oligonucleotide and were incubated at room temperature for 30 minutes. Binding reaction products were separated in a 4% polyacrylamide gel and analyzed by autoradiography.