PMC:1920263 / 13045-15338 JSON TXT

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test2

{"project":"test2","denotations":[{"id":"T4954","span":{"begin":789,"end":792},"obj":"Protein"},{"id":"T4955","span":{"begin":880,"end":883},"obj":"Protein"},{"id":"T4956","span":{"begin":1111,"end":1114},"obj":"Protein"},{"id":"T4957","span":{"begin":1263,"end":1287},"obj":"Protein"},{"id":"T4958","span":{"begin":1534,"end":1554},"obj":"Protein"},{"id":"T4959","span":{"begin":1995,"end":1998},"obj":"Protein"},{"id":"T4960","span":{"begin":2008,"end":2011},"obj":"Protein"},{"id":"T4961","span":{"begin":2107,"end":2110},"obj":"Protein"},{"id":"T4962","span":{"begin":2158,"end":2161},"obj":"Protein"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

GO-CC

{"project":"GO-CC","denotations":[{"id":"T5852","span":{"begin":97,"end":102},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5853","span":{"begin":287,"end":292},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5854","span":{"begin":2111,"end":2119},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T5855","span":{"begin":2162,"end":2170},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T5856","span":{"begin":2111,"end":2119},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T5857","span":{"begin":2162,"end":2170},"obj":"http://purl.obolibrary.org/obo/GO_0042571"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

GO-MF

{"project":"GO-MF","denotations":[{"id":"T5850","span":{"begin":2111,"end":2119},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T5851","span":{"begin":2162,"end":2170},"obj":"http://purl.obolibrary.org/obo/GO_0003823"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T4963","span":{"begin":1541,"end":1546},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

bionlp-st-ge-2016-spacy-parsed

sentences

pmc-enju-pas

events-check-again

{"project":"events-check-again","denotations":[{"id":"T5903","span":{"begin":789,"end":792},"obj":"Protein"},{"id":"T5904","span":{"begin":880,"end":883},"obj":"Protein"},{"id":"T5905","span":{"begin":1111,"end":1114},"obj":"Protein"},{"id":"T5906","span":{"begin":1263,"end":1287},"obj":"Protein"},{"id":"T5907","span":{"begin":1534,"end":1554},"obj":"Protein"},{"id":"T5908","span":{"begin":1995,"end":1998},"obj":"Protein"},{"id":"T5909","span":{"begin":2008,"end":2011},"obj":"Protein"},{"id":"T5910","span":{"begin":2107,"end":2110},"obj":"Protein"},{"id":"T5911","span":{"begin":2158,"end":2161},"obj":"Protein"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

bionlp-st-ge-2016-reference-tees

{"project":"bionlp-st-ge-2016-reference-tees","denotations":[{"id":"T5912","span":{"begin":785,"end":788},"obj":"Protein"},{"id":"T5913","span":{"begin":789,"end":792},"obj":"Protein"},{"id":"T5914","span":{"begin":876,"end":879},"obj":"Protein"},{"id":"T5915","span":{"begin":880,"end":883},"obj":"Protein"},{"id":"T5916","span":{"begin":1111,"end":1114},"obj":"Protein"},{"id":"T5917","span":{"begin":1146,"end":1153},"obj":"Protein"},{"id":"T5918","span":{"begin":1263,"end":1287},"obj":"Protein"},{"id":"T5919","span":{"begin":1534,"end":1554},"obj":"Protein"},{"id":"T5920","span":{"begin":1991,"end":2000},"obj":"Protein"},{"id":"T5921","span":{"begin":2004,"end":2007},"obj":"Protein"},{"id":"T5922","span":{"begin":2008,"end":2011},"obj":"Protein"},{"id":"T5923","span":{"begin":2012,"end":2016},"obj":"Protein"},{"id":"T5924","span":{"begin":2107,"end":2110},"obj":"Protein"},{"id":"T5925","span":{"begin":2158,"end":2161},"obj":"Protein"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

bionlp-st-ge-2016-reference

{"project":"bionlp-st-ge-2016-reference","denotations":[{"id":"T4964","span":{"begin":789,"end":792},"obj":"Protein"},{"id":"T4965","span":{"begin":880,"end":883},"obj":"Protein"},{"id":"T4966","span":{"begin":1111,"end":1114},"obj":"Protein"},{"id":"T4967","span":{"begin":1263,"end":1287},"obj":"Protein"},{"id":"T4968","span":{"begin":1534,"end":1554},"obj":"Protein"},{"id":"T4969","span":{"begin":1995,"end":1998},"obj":"Protein"},{"id":"T4970","span":{"begin":2008,"end":2011},"obj":"Protein"},{"id":"T4971","span":{"begin":2107,"end":2110},"obj":"Protein"},{"id":"T4972","span":{"begin":2158,"end":2161},"obj":"Protein"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

bionlp-st-ge-2016-uniprot

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T5369","span":{"begin":511,"end":513},"obj":"P0A7Z4"},{"id":"T5370","span":{"begin":789,"end":792},"obj":"P08047"},{"id":"T5371","span":{"begin":880,"end":883},"obj":"P08047"},{"id":"T5372","span":{"begin":1111,"end":1114},"obj":"P08047"},{"id":"T5373","span":{"begin":1995,"end":1998},"obj":"P08047"},{"id":"T5374","span":{"begin":2008,"end":2011},"obj":"P08047"},{"id":"T5375","span":{"begin":2107,"end":2110},"obj":"P08047"},{"id":"T5376","span":{"begin":2158,"end":2161},"obj":"Q02447"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"Electrophoretic mobility shift assay (EMSA)\nFor 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.\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 30 min."}

PMC:1920263 / 13045-15338 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:1920263 / 13045-15338 JSON TXT