Analyzing DNA Binding of AP-1 and NFκB

LN cells of three mice per group and nuclear extracts probed by electro-mobility shift assays to analyse DNA binding of AP-1 or NFκB, as previously described (D'Acquisto et al., 2007 (link)). Briefly, nuclear extracts (3–5 μg) were incubated with 2 μg of poly (dI:dC) in 20 μl of binding buffer with ³²P end-labelled, double-stranded oligonucleotide probes (5×10⁵ cpm), and fractionated on a 6% polyacrylamide gel (29:1 cross-linking ratio) in 0.5% TBE for 2.5 h at 150 volts. Double–stranded oligonucleotide probes were from Promega, UK. Supershift EMSA were performed as above, additionally using the following antibodies: anti-cFos, -FosB, -JunB, -JunD (all Cell Signaling Technology)