Electrophoretic mobility shift assay
In vitro translation of NRSF and Sp3 was performed using the TNT SP6 and T7 Quick Coupled Transcription/Translation System as per the manufacturer's protocol (Promega) and nuclear extracts from NS20Y cells were prepared as described previously (11). Double-stranded oligonucleotides containing a copy of the MOR NRSE/GC box (5′-CAGCAAGCATTCAGAACCATGGACAGCAGCGCCGGCCCAGGGA-3′) were synthesized and end-labeled using the Klenow fragment of DNA polymerase I in the presence of [γ-32P]dATP. Free nucleotides were separated by centrifugation through a G-25 column (Roche). The end-labeled DNA probes were incubated with in vitro translated NRSF or Sp3 or both and with nuclear extract from NS20Y cells. For competition analysis, a 100-fold molar excess of cold competitor oligonucleotide was added to the mixture before adding the probe. For supershift assay, antibodies or pre-immune serum were pre-incubated for 30 min on ice before adding the labeled probe. Radiolabeled probe was then added and the mixture was incubated for further 20 min on ice. Samples were subjected to electrophoresis on a native 4% polyacrylamide gel run in 0.5× TBE buffer for 2 h at 180 V. Monoclonal antibody against NRSF was obtained from Dr D. J. Anderson. Anti-IRF4 antibody was purchased from Santa Cruz Biotechnology.