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pmc-enju-pas

bionlp-st-ge-2016-coref

{"project":"bionlp-st-ge-2016-coref","denotations":[{"id":"T13716","span":{"begin":381,"end":394},"obj":"Antecedent"},{"id":"T13717","span":{"begin":629,"end":640},"obj":"Anaphor"},{"id":"T13718","span":{"begin":641,"end":656},"obj":"Antecedent"},{"id":"T13719","span":{"begin":658,"end":671},"obj":"Antecedent"},{"id":"T13720","span":{"begin":676,"end":689},"obj":"Antecedent"},{"id":"T13721","span":{"begin":892,"end":908},"obj":"Anaphor"},{"id":"T13722","span":{"begin":777,"end":787},"obj":"Antecedent"},{"id":"T13713","span":{"begin":334,"end":345},"obj":"Anaphor"},{"id":"T13714","span":{"begin":346,"end":361},"obj":"Antecedent"},{"id":"T13715","span":{"begin":363,"end":376},"obj":"Antecedent"},{"id":"T26503","span":{"begin":955,"end":958},"obj":"Anaphor"},{"id":"T26504","span":{"begin":940,"end":943},"obj":"Antecedent"}],"relations":[{"id":"R11143","pred":"boundBy","subj":"T13713","obj":"T13714"},{"id":"R11144","pred":"boundBy","subj":"T13713","obj":"T13715"},{"id":"R11145","pred":"boundBy","subj":"T13713","obj":"T13716"},{"id":"R11146","pred":"boundBy","subj":"T13717","obj":"T13718"},{"id":"R11147","pred":"boundBy","subj":"T13717","obj":"T13719"},{"id":"R11148","pred":"boundBy","subj":"T13717","obj":"T13720"},{"id":"R11149","pred":"boundBy","subj":"T13721","obj":"T13722"},{"id":"R20924","pred":"boundBy","subj":"T26503","obj":"T26504"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Tat increases the p65 affinity binding to DNA through association with p65\nWe tested whether Tat physically interacted with p65. HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts. The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A). Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B). These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.\nFigure 4. Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 µg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49–57)A, or p3xFLAG-Tat C(22,25,27)A (5 µg). FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 µl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49–57)A, GST-Tat C(22,25,27)A, or GST (5 µg) conjugated with Glutathione-Sepharose. Protein complexes were recovered by GST-pull down, separated by 12% SDS–PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 µg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC (1–318), or pRc/CMV-3xHA-p65ΔN (122–551) (5 µg). Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-κB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 µl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide. DNA/protein complexes were separated by 6% PAGE in 0.5 × TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.\nTo identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65ΔC (1–318)], or the RHD [p65ΔN (122–551)] (Figure 4C). Tat coimmunoprecipitated with p65ΔC (1–318), and not with p65ΔN (122–551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.\nTo address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-κB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA. Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E). The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-κB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F)."}

bionlp-st-ge-2016-spacy-parsed

GO-BP

{"project":"GO-BP","denotations":[{"id":"T14263","span":{"begin":555,"end":574},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T14264","span":{"begin":2788,"end":2803},"obj":"http://purl.obolibrary.org/obo/GO_2000144"},{"id":"T26938","span":{"begin":1386,"end":1396},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T26939","span":{"begin":2340,"end":2350},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T26940","span":{"begin":2220,"end":2222},"obj":"http://purl.obolibrary.org/obo/GO_0033968"}],"text":"Tat increases the p65 affinity binding to DNA through association with p65\nWe tested whether Tat physically interacted with p65. HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts. The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A). Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B). These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.\nFigure 4. Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 µg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49–57)A, or p3xFLAG-Tat C(22,25,27)A (5 µg). FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 µl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49–57)A, GST-Tat C(22,25,27)A, or GST (5 µg) conjugated with Glutathione-Sepharose. Protein complexes were recovered by GST-pull down, separated by 12% SDS–PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 µg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC (1–318), or pRc/CMV-3xHA-p65ΔN (122–551) (5 µg). Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-κB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 µl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide. DNA/protein complexes were separated by 6% PAGE in 0.5 × TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.\nTo identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65ΔC (1–318)], or the RHD [p65ΔN (122–551)] (Figure 4C). Tat coimmunoprecipitated with p65ΔC (1–318), and not with p65ΔN (122–551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.\nTo address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-κB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA. Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E). The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-κB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F)."}

GO-MF

GO-CC

{"project":"GO-CC","denotations":[{"id":"T14278","span":{"begin":134,"end":139},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T14279","span":{"begin":2695,"end":2700},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T26946","span":{"begin":990,"end":995},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T26947","span":{"begin":1795,"end":1800},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T26948","span":{"begin":1245,"end":1253},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T26949","span":{"begin":2032,"end":2040},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T26950","span":{"begin":2148,"end":2158},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T26951","span":{"begin":1245,"end":1253},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T26952","span":{"begin":2032,"end":2040},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T26953","span":{"begin":2148,"end":2158},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T26954","span":{"begin":1570,"end":1587},"obj":"http://purl.obolibrary.org/obo/GO_0043234"},{"id":"T26955","span":{"begin":2493,"end":2510},"obj":"http://purl.obolibrary.org/obo/GO_0043234"},{"id":"T26956","span":{"begin":2489,"end":2510},"obj":"http://purl.obolibrary.org/obo/GO_0001114"},{"id":"T26957","span":{"begin":2489,"end":2510},"obj":"http://purl.obolibrary.org/obo/GO_0032993"},{"id":"T26958","span":{"begin":2489,"end":2510},"obj":"http://purl.obolibrary.org/obo/GO_0097522"}],"text":"Tat increases the p65 affinity binding to DNA through association with p65\nWe tested whether Tat physically interacted with p65. HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts. The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A). Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49–57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B). These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.\nFigure 4. Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 µg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49–57)A, or p3xFLAG-Tat C(22,25,27)A (5 µg). FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 µl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49–57)A, GST-Tat C(22,25,27)A, or GST (5 µg) conjugated with Glutathione-Sepharose. Protein complexes were recovered by GST-pull down, separated by 12% SDS–PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 µg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC (1–318), or pRc/CMV-3xHA-p65ΔN (122–551) (5 µg). Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS–PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-κB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 µl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide. DNA/protein complexes were separated by 6% PAGE in 0.5 × TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.\nTo identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65ΔC (1–318)], or the RHD [p65ΔN (122–551)] (Figure 4C). Tat coimmunoprecipitated with p65ΔC (1–318), and not with p65ΔN (122–551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.\nTo address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-κB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA. Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E). The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-κB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F)."}

sentences

events-check-again

bionlp-st-ge-2016-reference-tees

test2

bionlp-st-ge-2016-reference

bionlp-st-ge-2016-uniprot

PMC:3333881 / 26459-30143 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:3333881 / 26459-30143 JSON TXT