Co-immunoprecipitation and western blots
Cells were lysed in a buffer containing 1% Triton X-100, 10 mM Tris, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, supplemented with 1 mg/ml antipain, 1 mg/ml leupeptin, 100 nM PMSF and 100 nM sodium orthovanadate. Approximately 1 mg of the clarified cell lysate was incubated overnight at 4°C with an anti-Sp1, anti-Sp3 or anti-NRSF monoclonal antibody. Immunoprecipitates were recovered on protein G–Sepharose beads, washed extensively and separated on SDS–PAGE. Proteins were transferred on to PVDF membrane (Amersham Biosciences). Membranes were incubated with anti-Sp3 (sc-644; Santa Cruz Biotechnology), anti-Sp1 (sc-59; Santa Cruz Biotechnology) and anti-NRSF antibody (12C11; kindly provided by Dr David Anderson) and signals were detected using a Storm 840 PhosphorImager system (Amersham Biosciences).