PMC:1920263 / 13045-15338 JSONTXT

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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.\nEMSA 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).\nFor 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 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