Electrophoretic mobility shift assay (EMSA)
For preparation of nuclear extracts, 5 × 106 A3.01 T cells were washed in cold PBS and resuspended in 500 µl buffer A (10 mM HEPES pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). After incubation for 15 min on ice, swollen cells were pressed 10 times through a syringe with a 26G needle and centrifuged at 5000 r.p.m. for 5 min. Pellets contained the nuclei and were washed in buffer A for two times and resuspended in 50 µl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA 1 mM DTT, 1 mM PMSF). After shaking for 30 min at 4°C and centrifugation for 10 min at 13 000 r.p.m., supernatants were used as nuclear extracts.
EMSA probes were generated by annealing the following complementary oligonucleotides: APO-Sp1/3, 5′- CCAGCTGGGCGGGACCACCAGGGGAGGGGC-3′ and 5′-GCCCCTCCCCTGGTGGTCCCGCCCAGCTGG-3′; APO-Sp1/3mut, 5′- CCAGCTGTTCGGGACCACCAGGGGAGGGGC-3′ and 5′- GCCCCTCCCCTGGTGGTCCCGAACAGCTGG-3′ according to standard procedures. Nucleotides differing from the original promoter sequence are shown in bold type. A commercially available Sp1 probe (sc-2502, referred to as Sp1cons) was purchased from Santa Cruz Biotechnology. The double-stranded oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (3000 Ci/mmol, Amersham) and purified by using Nick G50 columns (Amersham).
For EMSA, 5 µg of nuclear proteins were preincubated on ice with 2 µg of poly(dI-dC) (Roche) as an unspecific competitor and 1 µg of bovine serum albumin in band shift buffer (50 mM Tris, 150 mM KCl, 5 mM EDTA, 2.5 mM dithiothreitol, 20% Ficoll) for 15 min. 32P-labeled oligonucleotides (50 000 c.p.m.) were added in a total volume of 20 µl, incubated on ice for 20 min and loaded onto 5% native polyacrylamide gels in 0.5×Tris-borate-EDTA buffer. Upon fractionation, gels were dried and exposed for autoradiography. For competition experiments, 1- or 30-fold molar excess of the unlabeled APO-Sp1/3 or APO-Sp1/3mut oligonucleotides was added to the preincubation mixture. For supershift experiments, 2 µg Sp1 antibody (sc-59x, Santa Cruz Biotechnology) or Sp3 antibody (sc-644x, Santa Cruz Biotechnology) were added to the preincubation mixture and preincubation time was extended to 30 min.