32p datp

The shift assay was conducted with the NF-κB gel shift oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGGC-3′) obtained from Promega (Madison, WI, USA). The single-stranded oligonucleotides contained in the cellular extracts were end-labeled with [32P]-deoxyadenosine triphosphate (dATP) (Amersham Biosciences, Piscataway, NJ, USA) using T4 polynucleotide kinase (GIBCO). The radiolabeled oligonucleotides were separated from unincorporated [32P]-dATP by chromatography on a Bio-Rad purification column (Bio-Rad Laboratories) using Tris-EDTA buffer as eluant. Nuclear extracts were incubated with [32P]-radiolabeled probes in buffer (12% glycerol, 12 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, 25 mM KCl, 5 mM MgCl2, 0.04 µg/mL poly[d(I-C)]) for 30 min at room temperature. For supershift analysis, antibodies directed against p50 or p65 were added to the nuclear extract 30 min prior to the reaction. The samples were analyzed by electrophoretic separation at 4 °C on a nondenaturing 5% acrylamide gel. After drying at 80 °C for 2 h, the gel was exposed to a radiography film on intensifying screens for 6 to 18 h at −80 °C.

The NF-κB gel shift oligonucleotide (5’-ACTTGAGGGGACTTTCCCAGGGC-3’) and the PPAR gel shift oligonucleotide (5’-CAAAACTAGGTCAAAGCTCA-3’; sc-2587, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were radiolabeled using [³²P]-dATP (Amersham Biosciences, Piscataway, NJ, USA) and T4 polynucleotide kinase (GIBCO, Grand Island, NY, USA). The radiolabeled oligonucleotide was separated from unconsumed [³²P]-dATP using a Bio-Rad purification column (Bio-Rad Laboratories) eluted with Tris-EDTA buffer. Nuclear extracts of the cells were incubated with the [³²P]-labeled oligonucleotide in buffer containing 12% glycerol, 12 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, 25 mM KCl, 5 mM MgCl₂, 0.04 μg/mL poly[d(I-C)] at room temperature for 30 min. The samples were subjected to electrophoretic separation at 4 °C on a nondenaturing, 5% acrylamide gel. The gel was dried at 80 °C for 2 h after which it was exposed at −80 °C to a radiography film using intensifying screens.

A NF-κB gel shift oligonucleotide (AGTTGAGGGGACTTTCCCAGGC) and a AP-1 gel-shift oligonucleotide (CGCTTGATA GTCAGCCGGAA) (all from Promega, Madison, WI, USA) were labeled with [³²P] dATP (Amersham) using the T4 polynucleotide kinase (GIBCO, Grand Island, NY, USA). The end-labeled probe was purified from an unincorporated [³²P] dATP using a Bio-Rad purification column (Bio-Rad Laboratories) and recovered in Tris-EDTA buffer (TE). Nuclear extracts (3 μg) were incubated with the buffer containing ³²P-labeled NF-κB or AP-1 consensus oligonucleotide for 30 min and subjected to electrophoretic separation on a nondenaturing acrylamide gel. The gels were dried at 80°C for 2 h and exposed to a radiography film for 6–18 h at −70°C with intensifying screens [28 ].