Electrophoretic mobility shift assay (EMSA)
For preparation of nuclear extracts, 5 x 106 A3.01 T cells were washed in cold PBS and resuspended in 500 microl 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 microl 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 4degreesC 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 [gamma-32P]ATP (3000 Ci/mmol, Amersham) and purified by using Nick G50 columns (Amersham). 
For EMSA, 5 microg of nuclear proteins were preincubated on ice with 2 microg of poly(dI-dC) (Roche) as an unspecific competitor and 1 microg 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 microl, incubated on ice for 20 min and loaded onto 5% native polyacrylamide gels in 0.5xTris-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 microg 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. 
