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bionlp-st-ge-2016-reference

bionlp-st-ge-2016-reference-tees

events-check-again

pmc-enju-pas

2_test

bionlp-st-ge-2016-spacy-parsed

GO-BP

{"project":"GO-BP","denotations":[{"id":"T12839","span":{"begin":1701,"end":1708},"obj":"http://purl.obolibrary.org/obo/GO_0051923"},{"id":"T13298","span":{"begin":2418,"end":2430},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T13299","span":{"begin":2482,"end":2494},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T13507","span":{"begin":3852,"end":3854},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T13803","span":{"begin":3916,"end":3929},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T14231","span":{"begin":4494,"end":4497},"obj":"http://purl.obolibrary.org/obo/GO_0004705"},{"id":"T14232","span":{"begin":4539,"end":4548},"obj":"http://purl.obolibrary.org/obo/GO_0023052"},{"id":"T14717","span":{"begin":5906,"end":5911},"obj":"http://purl.obolibrary.org/obo/GO_0019835"},{"id":"T14718","span":{"begin":6036,"end":6047},"obj":"http://purl.obolibrary.org/obo/GO_0016791"}],"text":"Materials and Methods\n\nCell lines\nThe mouse B cell line A20.2J has been previously described [26], [27]. Cells were grown in RPMI 1640, 10% FCS, 10 µM 2-ME, 2 mM L-glutamine, and antibiotics. HI-5 insect cells (Invitrogen) expressing CD154 have been described [28].\n\nAntibodies\nMouse anti-birch profilin antibody (4A6 [29]), rat anti-mouse CD40 (1C10 [30]) and a rat isotype control antibody (mAb72, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) were isolated from hybridoma supernatants. FITC-labeled anti-mouse CD80 and an isotype control Ab were from eBiosciences. Rabbit anti-HOIP Ab [6] was the kind gift of Dr. Betty Eipper (University of Connecticut Health Center, Farmington, Connecticut). Goat anti-rat IgG, and HRP-labeled secondary Abs were from Jackson ImmunoResearch Laboratories, Inc. All other antibodies used were described previously [6].\n\nSomatic cell gene targeting\nThe generation of HOIP-deficient cells was accomplished using a homologous recombination approach described previously [7], [8]. Segments of Rnf31 gene sequence used in the targeting construct (Fig. 1A) were amplified by PCR from A20.2J genomic DNA. The oligonucleotide primers used to generate the 5′ flank (1224 bp) were 5′-ttttctagagcggtggcttaagtgaccc-3′ and 5′-tattctagatgcagcatctgagaaagcaagc-3′. The 3′ flank (6032 bp) primers were 5′-aaaaccggtgtatgcttctttacgggagaaaaatattag-3′ and 5′-tataccggtatgaagccaaaggaacactgagag-3′. Restriction endonuclease sites in the oligonucleotide primers allowed insertion of the PCR products into the targeting vector. A20.2J cells were transfected (by electroporation [7]) with the targeting construct and subcloned in medium containing 600 µg/ml G418 sulfate. Homologous recombination in G418-resistant clones was detected by PCR of genomic DNA, as described [7]. Oligonucleotide primers used for screening were 5′-cttcctgatctcagctttaccgtcac-3′ (homologous to genomic sequence; approximate position noted in Fig. 1) and 5′-caatccatcttgttcagccat-3′ (homologous to sequence in NeoR). Clones in which one copy of Rnf31 had been disrupted were transiently transfected with an expression plasmid encoding Cre, in order to remove NeoR. G418-sensitive subclones were subjected to a second round of targeting to disrupt the remaining copy of Rnf31. G418-resistant clones were tested for homologous recombination by PCR and HOIP protein expression by Western blot.\n\nRetroviral transduction\nA retroviral vector (pMIP) was used for the stable transduction of cells with HOIP and IKKγ cDNA constructs. pMIP was constructed by replacing the IRES and GFP encoding sequences in pMIG [31] with the IRES and puromycin resistance gene from pIRESpuro2 (Clontech). FLAG-tagged mouse HOIP cDNA was inserted into the multiple cloning site of pMIP. A pMIP construct encoding mouse IKKγ with an amino terminal birch profilin peptide (BP)-tag was also prepared. Retroviral particles were generated by transient transfection of 293T cells with pMIP, pMIP-HOIP, or pMIP-IKKγ and the packaging vector pCL-Eco [32]. Two days after transfection, culture supernatants were harvested and filtered. A20.2J or HOIP-deficient cells were added to 24-well plates (2.5×105 cells/well) with 1 ml virus-containing supernatant and 8 µg/ml polybrene. Plates were centrifuged at 500 x g for 2 hrs at room temperature. Cells were cultured in B cell culture medium for 48 hrs, after which the transduced cells were selected in 3 µg/ml puromycin.\n\nCD80 upregulation and flow cytometry\nCells (5×104 in 2 ml) were cultured in 24-well plates for 3 days with 5 µg/ml anti-CD40 or an isotype control antibody. Cells were stained for flow cytometry with FITC-anti-CD80 or an appropriate control antibody as described [28]. Data acquired with a FACScan flow cytometer (BD Biosciences) were analyzed with WinMDI 2.8 (Scripps Research Institute, San Diego, CA).\n\nGLε transcript assay\nThe CD40-simulated activation of GLε transcription was evaluated as previously reported [33]. Briefly, 1×106 cells were stimulated overnight with anti-CD40 antibody (10 µg/ml) or an isotype control antibody, with or without 500 U/ml mouse IL-4 (BD Biosciences). RNA was isolated using Trizol (Invitrogen), and reverse-transcribed (Superscript III kit, Invitrogen). Quantitative PCR for GLε and Hprt1 was performed using SYBR GREEN master mix (Applied Biosystems), and an Applied Biosystems 7900HT Fast Real-Time PCR instrument. Expression of GLε in each sample was normalized to the expression of Hprt1.\n\nNF-κB and JNK activation assays\nFor activation of CD40 signaling, 5×104 HI5 insect cells or HI5 insect cells expressing mouse CD154 (CD40 ligand) were added to 1×106 B cells. Cells were centrifuged for 1 minute at 400 x g (to promote contact between ligand cells and B cells) then incubated at 37°C for times indicated in Fig. 4. Following stimulation, cells were chilled on ice for 2 minutes. Cell pellets were dissolved in 2X SDS-PAGE loading buffer, sonicated, and heated for 5 minutes at 95°C. Total cell lysates (1×105 cell equivalents per lane) were fractionated by SDS-PAGE and blotted to polyvinylidine fluoride (PVDF) membranes. Membranes were probed with antibodies as indicated in Fig. 4. Chemiluminescent detection (Pierce Biotechnology) was used for the visualization of bands on Western blots. Images of blots were recorded with a low-light imaging system (LAS4000, Fuji Medical Systems) and on X-ray film.\n\nImmunoprecipitations\nImmunoprecipitation of CD40 was performed by activated receptor capture, as previously described [6]. Briefly, 3×107 cells (A20.2J and derivatives) were incubated for 60 minutes at room temperature with 10 µl magnetic protein G beads (Dynal) pre-coated with 10 µg goat anti-rat IgG (Jackson), and 10 µg anti-CD40 (1C10) or an isotype control antibody (mAb72). Cells/beads were then pelleted by centrifugation and lysed in buffer containing 1% Triton X100. Beads were washed with lysis buffer to remove unbound material. In some experiments, material associated with the beads was dephosphorylated with lambda phosphatase (New England BioLabs) as per manufacturer's instructions. Beads were resuspended in 2X SDS-PAGE sample buffer and heated for 5 minutes at 95°C. Material eluted from the beads was fractionated by SDS-PAGE and transferred to PVDF membranes for Western blotting. In some experiments, cells were cultured for 6 hrs with 25 µM antennapedia-linked SMAC-N7 peptide (Calbiochem) or an appropriate volume of the solvent used for the peptide (DMSO). After incubation, cells (in peptide- or DMSO-containing medium) were stimulated with antibody-coated beads as outlined above. "}

GO-MF

{"project":"GO-MF","denotations":[{"id":"T12281","span":{"begin":304,"end":312},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T12282","span":{"begin":383,"end":391},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T12283","span":{"begin":836,"end":846},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T13508","span":{"begin":3599,"end":3607},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T13509","span":{"begin":3693,"end":3701},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T13510","span":{"begin":3852,"end":3854},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T13804","span":{"begin":4035,"end":4043},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T13805","span":{"begin":4077,"end":4085},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T13806","span":{"begin":4118,"end":4122},"obj":"http://purl.obolibrary.org/obo/GO_0005136"},{"id":"T14233","span":{"begin":4494,"end":4497},"obj":"http://purl.obolibrary.org/obo/GO_0004705"},{"id":"T14234","span":{"begin":4622,"end":4628},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T14235","span":{"begin":4734,"end":4740},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T14236","span":{"begin":5149,"end":5159},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T14719","span":{"begin":5769,"end":5777},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T14720","span":{"begin":6573,"end":6581},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T14721","span":{"begin":6036,"end":6047},"obj":"http://purl.obolibrary.org/obo/GO_0016791"}],"text":"Materials and Methods\n\nCell lines\nThe mouse B cell line A20.2J has been previously described [26], [27]. Cells were grown in RPMI 1640, 10% FCS, 10 µM 2-ME, 2 mM L-glutamine, and antibiotics. HI-5 insect cells (Invitrogen) expressing CD154 have been described [28].\n\nAntibodies\nMouse anti-birch profilin antibody (4A6 [29]), rat anti-mouse CD40 (1C10 [30]) and a rat isotype control antibody (mAb72, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) were isolated from hybridoma supernatants. FITC-labeled anti-mouse CD80 and an isotype control Ab were from eBiosciences. Rabbit anti-HOIP Ab [6] was the kind gift of Dr. Betty Eipper (University of Connecticut Health Center, Farmington, Connecticut). Goat anti-rat IgG, and HRP-labeled secondary Abs were from Jackson ImmunoResearch Laboratories, Inc. All other antibodies used were described previously [6].\n\nSomatic cell gene targeting\nThe generation of HOIP-deficient cells was accomplished using a homologous recombination approach described previously [7], [8]. Segments of Rnf31 gene sequence used in the targeting construct (Fig. 1A) were amplified by PCR from A20.2J genomic DNA. The oligonucleotide primers used to generate the 5′ flank (1224 bp) were 5′-ttttctagagcggtggcttaagtgaccc-3′ and 5′-tattctagatgcagcatctgagaaagcaagc-3′. The 3′ flank (6032 bp) primers were 5′-aaaaccggtgtatgcttctttacgggagaaaaatattag-3′ and 5′-tataccggtatgaagccaaaggaacactgagag-3′. Restriction endonuclease sites in the oligonucleotide primers allowed insertion of the PCR products into the targeting vector. A20.2J cells were transfected (by electroporation [7]) with the targeting construct and subcloned in medium containing 600 µg/ml G418 sulfate. Homologous recombination in G418-resistant clones was detected by PCR of genomic DNA, as described [7]. Oligonucleotide primers used for screening were 5′-cttcctgatctcagctttaccgtcac-3′ (homologous to genomic sequence; approximate position noted in Fig. 1) and 5′-caatccatcttgttcagccat-3′ (homologous to sequence in NeoR). Clones in which one copy of Rnf31 had been disrupted were transiently transfected with an expression plasmid encoding Cre, in order to remove NeoR. G418-sensitive subclones were subjected to a second round of targeting to disrupt the remaining copy of Rnf31. G418-resistant clones were tested for homologous recombination by PCR and HOIP protein expression by Western blot.\n\nRetroviral transduction\nA retroviral vector (pMIP) was used for the stable transduction of cells with HOIP and IKKγ cDNA constructs. pMIP was constructed by replacing the IRES and GFP encoding sequences in pMIG [31] with the IRES and puromycin resistance gene from pIRESpuro2 (Clontech). FLAG-tagged mouse HOIP cDNA was inserted into the multiple cloning site of pMIP. A pMIP construct encoding mouse IKKγ with an amino terminal birch profilin peptide (BP)-tag was also prepared. Retroviral particles were generated by transient transfection of 293T cells with pMIP, pMIP-HOIP, or pMIP-IKKγ and the packaging vector pCL-Eco [32]. Two days after transfection, culture supernatants were harvested and filtered. A20.2J or HOIP-deficient cells were added to 24-well plates (2.5×105 cells/well) with 1 ml virus-containing supernatant and 8 µg/ml polybrene. Plates were centrifuged at 500 x g for 2 hrs at room temperature. Cells were cultured in B cell culture medium for 48 hrs, after which the transduced cells were selected in 3 µg/ml puromycin.\n\nCD80 upregulation and flow cytometry\nCells (5×104 in 2 ml) were cultured in 24-well plates for 3 days with 5 µg/ml anti-CD40 or an isotype control antibody. Cells were stained for flow cytometry with FITC-anti-CD80 or an appropriate control antibody as described [28]. Data acquired with a FACScan flow cytometer (BD Biosciences) were analyzed with WinMDI 2.8 (Scripps Research Institute, San Diego, CA).\n\nGLε transcript assay\nThe CD40-simulated activation of GLε transcription was evaluated as previously reported [33]. Briefly, 1×106 cells were stimulated overnight with anti-CD40 antibody (10 µg/ml) or an isotype control antibody, with or without 500 U/ml mouse IL-4 (BD Biosciences). RNA was isolated using Trizol (Invitrogen), and reverse-transcribed (Superscript III kit, Invitrogen). Quantitative PCR for GLε and Hprt1 was performed using SYBR GREEN master mix (Applied Biosystems), and an Applied Biosystems 7900HT Fast Real-Time PCR instrument. Expression of GLε in each sample was normalized to the expression of Hprt1.\n\nNF-κB and JNK activation assays\nFor activation of CD40 signaling, 5×104 HI5 insect cells or HI5 insect cells expressing mouse CD154 (CD40 ligand) were added to 1×106 B cells. Cells were centrifuged for 1 minute at 400 x g (to promote contact between ligand cells and B cells) then incubated at 37°C for times indicated in Fig. 4. Following stimulation, cells were chilled on ice for 2 minutes. Cell pellets were dissolved in 2X SDS-PAGE loading buffer, sonicated, and heated for 5 minutes at 95°C. Total cell lysates (1×105 cell equivalents per lane) were fractionated by SDS-PAGE and blotted to polyvinylidine fluoride (PVDF) membranes. Membranes were probed with antibodies as indicated in Fig. 4. Chemiluminescent detection (Pierce Biotechnology) was used for the visualization of bands on Western blots. Images of blots were recorded with a low-light imaging system (LAS4000, Fuji Medical Systems) and on X-ray film.\n\nImmunoprecipitations\nImmunoprecipitation of CD40 was performed by activated receptor capture, as previously described [6]. Briefly, 3×107 cells (A20.2J and derivatives) were incubated for 60 minutes at room temperature with 10 µl magnetic protein G beads (Dynal) pre-coated with 10 µg goat anti-rat IgG (Jackson), and 10 µg anti-CD40 (1C10) or an isotype control antibody (mAb72). Cells/beads were then pelleted by centrifugation and lysed in buffer containing 1% Triton X100. Beads were washed with lysis buffer to remove unbound material. In some experiments, material associated with the beads was dephosphorylated with lambda phosphatase (New England BioLabs) as per manufacturer's instructions. Beads were resuspended in 2X SDS-PAGE sample buffer and heated for 5 minutes at 95°C. Material eluted from the beads was fractionated by SDS-PAGE and transferred to PVDF membranes for Western blotting. In some experiments, cells were cultured for 6 hrs with 25 µM antennapedia-linked SMAC-N7 peptide (Calbiochem) or an appropriate volume of the solvent used for the peptide (DMSO). After incubation, cells (in peptide- or DMSO-containing medium) were stimulated with antibody-coated beads as outlined above. "}

GO-CC

sentences

bionlp-st-ge-2016-uniprot

test2

PMC:3148254 / 27686-34300 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:3148254 / 27686-34300 JSON TXT