Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from control or treated cells were prepared as
previously described (Kinoshita, Yshii et al., 2017). Doublestranded
oligonucleotide containing the NF-κB consensus sequence from Promega
(5′-AGTTGAGGGGACTTTCCCAGGC-3′) was end labelled using T4 polynucleotide
kinase (Promega) in the presence of γ-32P dATP. Nuclear extracts (2.5
μg) were incubated with 32P-labelled NF-κB probe. The binding reaction
was performed at room temperature for 30 min in a reaction buffer
containing 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM MgCl2, 2.5 mM EDTA,
20 % glycerol, 0.25 μg/μL of poly (dI-dC) and 2.5 mM dithiothreitol.
DNA protein complexes were separated by electrophoresis through a 6 %
acrylamide:bis-acrylamide (37.5:1) gel in TBE (45 mM Tris, 45 mM boric
acid, 0.5 mM EDTA) for 2 h at 150 V. Gels were vacuum dried for 1 h at
80 °C and exposed to X-ray film at −80 °C. For competition assays,
nuclear extract was incubated with specific competitor (unlabelled
double-stranded NF-κB consensus oligonucleotide) or a non-specific
competitor (unlabelled transcription initiation factor IID [TFIID]).
For supershift assay, antibodies against subunits of NF-κB (p50 and
p65,1:20) (Santa Cruz Biotechnology) were added into the binding
reactions. Autoradiographs were visualized using a photodocumentation
system DP-001-FDC and quantified with ImageJ (NIH) software 70.