Nf κb gel shift oligonucleotide probe

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 shift assay was performed using an NF-κB gel shift oligonucleotide probe (5′-ACTTGAGGGGACTTTCCCAGGGC-3′) obtained from Promega (Madison, WI, USA). An aliquot of the single-stranded oligonucleotides was end-labeled with [³²P]-deoxyadenosine triphosphate (dATP) (Amersham Biosciences, Piscataway, NJ, USA) using T4 polynucleotide kinase (GIBCO, Grand Island, NY, USA). The radiolabeled oligonucleotides were separated from unincorporated [³²P] dATP using the Bio-Rad purification column (Bio-Rad Laboratories), and were purified with tris-EDTA buffer. Nuclear extracts isolated from the cells 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 21–23 °C. 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 and exposed to a radiography film at −80 °C, with intensifying screens.