Gel shift assay system

EMSA was performed using a gel shift assay system (Promega). Nuclear protein extracts from HFK18 cells were prepared as described above. HeLa nuclear extract was included in the gel shift assay system as a positive control. The double-stranded oligonucleotides used as probes were 5′ end labeled with [γ-³²]pATP by T4 polynucleotide kinase and purified using Illustra MicroSpin G-25 columns (GE Healthcare). The oligonucleotides used as probes and as cold competitors are shown in Table S2. Nuclear extracts were incubated with gel shift binding buffer in the presence or absence of a cold competitor oligonucleotide and incubated at room temperature for 10 min, and then ³²P-labeled double-stranded oligonucleotide probes were added for an additional 20 min of incubation at room temperature. After 1 µl of room-temperature gel loading, 10× buffer was added per reaction and the reaction mixture was electrophoresed through a 4% polyacrylamide gel in 0.5× Tris-borate-EDTA (TBE) buffer, first at 200 V for 10 min and then at 350 V for an additional 50 min. The gel was then dried and exposed to a PhophorImager screen. The image was captured using a Molecular Dynamic PhosphorImager Storm 860 system and analyzed with ImageQuant software.

NF-κB was examined using electrophoretic mobility shift assay (EMSA). Nuclear extracts of BM-MSCs were prepared by hypotonic lysis followed by high salt extraction. EMSA was performed with the Gel Shift assay system (Promega, Madison, WI). In a typical experiment, The NF-κB consensus oligonucleotide probe (5′- AGT TGA GGG GAC TTT CCC AGG C -3′) (Promega) end-radiolabeled with [γ-32P]ATP (3000 Ci/mmol; Perkin-Elmer Life Technology, Waltham, MA) and T4 polynucleotide kinase (Promega) were incubated with nuclear extract, 100 µg/ml poly dI-dC, 10 mM Tris/HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 1 mM MgCl₂, and 4% glycerol according to the manufacturer’s instructions. After the incubation, samples were charged on 4% native polyacrylamide gels with a 0.5 × TBE running buffer and visualized by autoradiography. A 100-fold molar excess of unlabeled competitor was added to the reaction mixture before adding the nuclear extracts in some experiments. BAY11-7082 (Sigma, 5 µM) and pCMV-IκBα-M, a dominant-negative form of IκBα (Clontech, Mountain View, CA) was used for inhibition of NF-κB activation.

Cells were washed and scraped in ice-cold PBS to prepare nuclei for electrophoretic gel mobility shift assay with the use of the gel shift assay system modified according to the manufacturer's instructions (Promega). In brief, consensus oligonucleotides for damage or repair DNA was biotin-labeled (hot probe). Each binding reaction was carried out with 1 μg biotinylated dsDNA probe and 200 μg purified nuclear protein in 20 μl of binding buffer containing 0.5mg/ml poly (dI:dC) (25 mM HEPES at PH8.0 with 50 mM KCl. 0.1% Triton X100, 2 mM MgCl2, 3 mM DTT, and 5% glycerol). Twenty-five pmol unlabeled cold DNA motifs (a 250-fold excess) were added in the competition assays. Reactions were carried out for 30 min incubation at room temperature, followed by overnight incubation at 4°C. Reaction mixtures were loaded onto 6% TBE polyacrylamide gels and separated in 0.5×TBE at 100v on ice until the dye front migrated two-thirds of the way to NC membranes and Western blotting for anti-biotin.