PMC:1920263 / 15340-17307 JSON TXT

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test2

{"project":"test2","denotations":[{"id":"T5934","span":{"begin":464,"end":484},"obj":"Protein"},{"id":"T5935","span":{"begin":778,"end":781},"obj":"Protein"},{"id":"T5936","span":{"begin":795,"end":798},"obj":"Protein"},{"id":"T5937","span":{"begin":815,"end":820},"obj":"Protein"},{"id":"T5938","span":{"begin":1209,"end":1212},"obj":"Protein"},{"id":"T5939","span":{"begin":1456,"end":1470},"obj":"Protein"},{"id":"T5940","span":{"begin":1683,"end":1686},"obj":"Protein"},{"id":"T5941","span":{"begin":1876,"end":1879},"obj":"Protein"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

GO-CC

{"project":"GO-CC","denotations":[{"id":"T6743","span":{"begin":0,"end":9},"obj":"http://purl.obolibrary.org/obo/GO_0000785"},{"id":"T6744","span":{"begin":649,"end":658},"obj":"http://purl.obolibrary.org/obo/GO_0000785"},{"id":"T6745","span":{"begin":49,"end":54},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6746","span":{"begin":509,"end":514},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6747","span":{"begin":753,"end":758},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6748","span":{"begin":768,"end":776},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T6749","span":{"begin":768,"end":776},"obj":"http://purl.obolibrary.org/obo/GO_0042571"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

GO-MF

{"project":"GO-MF","denotations":[{"id":"T6742","span":{"begin":768,"end":776},"obj":"http://purl.obolibrary.org/obo/GO_0003823"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

GO-BP

{"project":"GO-BP","denotations":[{"id":"T5972","span":{"begin":242,"end":247},"obj":"http://purl.obolibrary.org/obo/GO_0019835"},{"id":"T5973","span":{"begin":429,"end":438},"obj":"http://purl.obolibrary.org/obo/GO_0007586"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T5942","span":{"begin":519,"end":523},"obj":"http://purl.obolibrary.org/obo/UBERON_0000025"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

bionlp-st-ge-2016-spacy-parsed

sentences

pmc-enju-pas

events-check-again

{"project":"events-check-again","denotations":[{"id":"T6790","span":{"begin":464,"end":484},"obj":"Protein"},{"id":"T6791","span":{"begin":778,"end":781},"obj":"Protein"},{"id":"T6792","span":{"begin":795,"end":798},"obj":"Protein"},{"id":"T6793","span":{"begin":815,"end":820},"obj":"Protein"},{"id":"T6794","span":{"begin":1209,"end":1212},"obj":"Protein"},{"id":"T6795","span":{"begin":1456,"end":1470},"obj":"Protein"},{"id":"T6796","span":{"begin":1683,"end":1686},"obj":"Protein"},{"id":"T6797","span":{"begin":1876,"end":1879},"obj":"Protein"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

bionlp-st-ge-2016-reference-tees

{"project":"bionlp-st-ge-2016-reference-tees","denotations":[{"id":"T6798","span":{"begin":0,"end":9},"obj":"Protein"},{"id":"T6799","span":{"begin":10,"end":29},"obj":"Binding"},{"id":"T6800","span":{"begin":332,"end":337},"obj":"Protein"},{"id":"T6801","span":{"begin":649,"end":658},"obj":"Protein"},{"id":"T6802","span":{"begin":763,"end":776},"obj":"Protein"},{"id":"T6803","span":{"begin":778,"end":781},"obj":"Protein"},{"id":"T6804","span":{"begin":782,"end":786},"obj":"Protein"},{"id":"T6805","span":{"begin":795,"end":798},"obj":"Protein"},{"id":"T6806","span":{"begin":799,"end":803},"obj":"Protein"},{"id":"T6807","span":{"begin":815,"end":820},"obj":"Protein"},{"id":"T6808","span":{"begin":1209,"end":1221},"obj":"Protein"},{"id":"T6809","span":{"begin":1456,"end":1470},"obj":"Protein"},{"id":"T6810","span":{"begin":1683,"end":1691},"obj":"Protein"},{"id":"T6811","span":{"begin":1876,"end":1897},"obj":"Protein"}],"relations":[{"id":"R5651","pred":"themeOf","subj":"T6798","obj":"T6799"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

bionlp-st-ge-2016-reference

{"project":"bionlp-st-ge-2016-reference","denotations":[{"id":"T5943","span":{"begin":464,"end":484},"obj":"Protein"},{"id":"T5944","span":{"begin":778,"end":781},"obj":"Protein"},{"id":"T5945","span":{"begin":795,"end":798},"obj":"Protein"},{"id":"T5946","span":{"begin":815,"end":820},"obj":"Protein"},{"id":"T5947","span":{"begin":1209,"end":1212},"obj":"Protein"},{"id":"T5948","span":{"begin":1456,"end":1470},"obj":"Protein"},{"id":"T5949","span":{"begin":1683,"end":1686},"obj":"Protein"},{"id":"T5950","span":{"begin":1876,"end":1879},"obj":"Protein"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

bionlp-st-ge-2016-uniprot

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T6306","span":{"begin":370,"end":372},"obj":"P0A7Z4"},{"id":"T6307","span":{"begin":778,"end":781},"obj":"P08047"},{"id":"T6308","span":{"begin":795,"end":798},"obj":"Q02447"},{"id":"T6309","span":{"begin":1209,"end":1212},"obj":"Q9HC16"},{"id":"T6310","span":{"begin":1683,"end":1686},"obj":"Q9HC16"},{"id":"T6311","span":{"begin":1876,"end":1879},"obj":"Q9HC16"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37°C. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 × 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 µl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5′-ccacggtggcctccgagggtga-3′ and ChIP3Gminus: 5′-ctctccaccatcaagacagac-3′ (1. PCR); ChIP3G2plus: 5′-tactctccctccctgtcccca-3′ and ChIP3G nested minus: 5′-aggctgatgcctccgcag-3′ (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5′-taagtaccacccagagatgag-3′ and ChIP3Gneg_minus: 5′-catgatcttcatggtggcacg-3′ for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55°C."}

PMC:1920263 / 15340-17307 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:1920263 / 15340-17307 JSON TXT