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PMC:3148254 JSONTXT 20 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T890 0-27 Protein denotes HOIL-1L Interacting Protein
T891 29-33 Protein denotes HOIP
T892 52-56 Protein denotes CD40
T893 115-119 Protein denotes CD40
T896 264-274 Binding denotes engagement
T894 278-282 Protein denotes CD40
T895 297-302 Protein denotes CD154
T897 412-416 Protein denotes CD40
T898 530-534 Protein denotes CD40
T901 535-545 Binding denotes engagement
T902 618-628 Positive_regulation denotes activation
T903 618-628 Positive_regulation denotes activation
T899 632-637 Protein denotes NF-κB
T900 639-671 Protein denotes stress-activated protein kinases
T904 738-742 Protein denotes CD40
T905 833-837 Protein denotes CD40
T906 879-909 Protein denotes TNF receptor-associated factor
T907 911-915 Protein denotes TRAF
T908 967-971 Protein denotes TRAF
T909 986-991 Protein denotes TRAF2
T915 1001-1012 Binding denotes recruitment
T916 1001-1012 Binding denotes recruitment
T917 1001-1012 Binding denotes recruitment
T918 1001-1012 Binding denotes recruitment
T910 1016-1043 Protein denotes HOIL-1L-interacting protein
T911 1045-1049 Protein denotes HOIP
T914 1070-1076 Entity denotes domain
T912 1080-1084 Protein denotes CD40
T913 1125-1129 Protein denotes CD40
T919 1174-1178 Protein denotes HOIP
T920 1182-1186 Protein denotes CD40
T921 1302-1306 Protein denotes CD40
T922 1331-1335 Protein denotes CD80
T925 1340-1350 Positive_regulation denotes activation
T923 1354-1377 Protein denotes germline immunoglobulin
T924 1386-1399 Transcription denotes transcription
T926 1463-1467 Protein denotes CD40
T929 1477-1487 Positive_regulation denotes activation
T930 1477-1487 Positive_regulation denotes activation
T927 1491-1496 Protein denotes NF-κB
T928 1501-1513 Protein denotes c-Jun kinase
T931 1518-1526 Negative_regulation denotes impaired
T932 1518-1526 Negative_regulation denotes impaired
T936 1528-1539 Binding denotes Recruitment
T933 1543-1562 Protein denotes IκB kinase proteins
T934 1570-1574 Protein denotes CD40
T935 1672-1677 Protein denotes NF-κB
T937 1678-1688 Positive_regulation denotes activation
T938 1705-1709 Protein denotes HOIP
T939 1710-1720 Gene_expression denotes expression
T940 1832-1836 Protein denotes CD40
T2561 1870-1874 Protein denotes CD40
T2562 2082-2086 Protein denotes CD40
T2565 2100-2109 Positive_regulation denotes activated
T2563 2158-2162 Protein denotes CD40
T2564 2164-2169 Protein denotes CD154
T2566 2174-2183 Gene_expression denotes expressed
T2567 2174-2183 Gene_expression denotes expressed
T2568 2216-2220 Protein denotes CD40
T2571 2221-2231 Binding denotes engagement
T2569 2298-2330 Protein denotes stress-activated protein kinases
T2570 2339-2365 Protein denotes transcription factor NF-κB
T2572 2484-2488 Protein denotes CD40
T2573 2581-2585 Protein denotes CD40
T2574 2747-2777 Protein denotes TNF receptor-associated factor
T2575 2779-2783 Protein denotes TRAF
T2581 2785-2791 Entity denotes family
T2576 2803-2808 Protein denotes TRAF1
T2577 2810-2815 Protein denotes TRAF2
T2578 2817-2822 Protein denotes TRAF3
T2579 2828-2833 Protein denotes TRAF6
T2582 2861-2870 Regulation denotes important
T2583 2861-2870 Regulation denotes important
T2584 2861-2870 Regulation denotes important
T2585 2861-2870 Regulation denotes important
T2586 2861-2870 Regulation denotes important
T2587 2861-2870 Regulation denotes important
T2588 2861-2870 Regulation denotes important
T2589 2861-2870 Regulation denotes important
T2580 2908-2912 Protein denotes CD40
T2591 2982-2989 Binding denotes binding
T2590 3017-3021 Protein denotes CD40
T2592 3100-3105 Protein denotes TRAFs
T2593 3123-3125 Protein denotes E3
T2594 3126-3143 Protein denotes ubiquitin ligases
T2595 3245-3250 Protein denotes TRAFs
T2600 3256-3265 Binding denotes associate
T2596 3271-3275 Protein denotes CD40
T2597 3277-3282 Protein denotes TRAF3
T2598 3340-3345 Protein denotes TRAF2
T2599 3350-3355 Protein denotes TRAF6
T2601 3447-3474 Protein denotes HOIL-1L interacting protein
T2602 3476-3480 Protein denotes HOIP
T2603 3485-3501 Protein denotes ubiquitin ligase
T2607 3572-3581 Binding denotes recruited
T2608 3572-3581 Binding denotes recruited
T2609 3572-3581 Binding denotes recruited
T2604 3585-3589 Protein denotes CD40
T2605 3595-3600 Protein denotes TRAF2
T2610 3628-3638 Binding denotes engagement
T2606 3642-3646 Protein denotes CD40
T2611 3755-3760 Protein denotes TRAF2
T2612 3768-3772 Protein denotes CD40
T2615 3808-3817 Positive_regulation denotes necessary
T8615 3808-3817 Positive_regulation denotes necessary
T2614 3826-3836 Positive_regulation denotes activation
T2613 3840-3845 Protein denotes NF-κB
T2616 4044-4055 Protein denotes B cell CD40
T2617 4099-4103 Protein denotes CD40
T2618 4128-4132 Protein denotes CD80
T2619 4226-4230 Protein denotes CD40
T2620 4235-4239 Protein denotes IL-4
T2623 4247-4257 Gene_expression denotes production
T2624 4247-4257 Gene_expression denotes production
T2625 4247-4257 Gene_expression denotes production
T2621 4291-4331 Protein denotes immunoglobulin epsilon heavy chain locus
T2622 4356-4375 Protein denotes immunoglobulin gene
T2626 4474-4478 Protein denotes CD40
T2630 4488-4498 Positive_regulation denotes activation
T2633 4488-4498 Positive_regulation denotes activation
T2627 4502-4507 Protein denotes NF-κB
T2631 4516-4532 Positive_regulation denotes stress-activated
T2628 4533-4560 Protein denotes protein kinase c-Jun kinase
T2629 4562-4565 Protein denotes JNK
T2632 4571-4580 Negative_regulation denotes defective
T2634 4631-4636 Protein denotes NF-κB
T2638 4637-4647 Positive_regulation denotes activation
T2639 4649-4660 Binding denotes association
T2640 4649-4660 Binding denotes association
T2641 4649-4660 Binding denotes association
T8614 4649-4660 Binding denotes association
T2635 4668-4678 Protein denotes IκB kinase
T8613 4668-13430 Protein denotes IκB kinase (IKK) complex with CD40 was undetectable in HOIP-deficient cells. Together, our results indicate that HOIP plays a critical role in the activation of signaling pathways that regulate cellular responses to CD40 engagement. Results Generation of HOIP-deficient B cells via targeted disruption of Rnf31, the gene encoding HOIP In our previous study, we demonstrated that HOIP is recruited to the CD40 signaling complex in two mouse B cell lines [6]. Similar results were obtained with mouse splenocytes, indicating that the interaction of CD40 with HOIP is not limited to transformed B cell lines (unpublished observations). We also found that the recruitment of HOIP to CD40 was TRAF2-dependent and that overexpression of a truncated HOIP mutant partially inhibited CD40-mediated NF-κB activation. These results support the hypothesis that HOIP plays a role in CD40 signal transduction. To further define the role and evaluate the importance of HOIP in CD40 signaling, we used somatic cell gene targeting to disrupt the gene encoding HOIP in the mouse B cell line A20.2J. This cell line has been particularly useful in the characterization of CD40 signaling mechanisms due to the relative ease with which its genome can be modified by homologous recombination [7], [8], [9]. We used a targeting vector capable of undergoing homologous recombination with Rnf31 (the gene encoding HOIP) to disrupt the coding sequence of the gene in exon 5 (Fig. 1A). Following introduction of the vector, the neomycin-resistant clones that arose were screened by PCR amplification of genomic DNA to identify cells containing a disrupted Rnf31 allele. To remove the selectable marker gene cassette from the disrupted Rnf31 allele, recombinant cell lines were transiently transfected with a plasmid that encodes Cre recombinase. This step allowed us to perform a second round of targeting and drug selection, generating cells in which both copies of Rnf31 were disrupted. Two independent clonal cell lines were chosen for further analysis. HOIP protein expression was undetectable in both cell lines, as determined by Western blot analysis of cell lysates (Fig. 1B), demonstrating that the targeting process was successful. In the text that follows, the two gene-targeted cell lines are referred to as HOIP-deficient cells. 10.1371/journal.pone.0023061.g001 Figure 1 HOIP gene targeting. (A) Regions of Rnf31 gene sequence (bottom) used in the HOIP gene targeting vector (top) are shown. Arrows (F and R) indicate approximate positions of sequences homologous to oligonucleotides used for PCR-mediated detection of homologous recombination. Homologous recombination of the vector with Rnf31 resulted in a small chromosomal deletion and the in-frame insertion of neomycin phosphotransferase (NeoR) into the amino-terminal HOIP coding sequence. A diphtheria toxin (DT) cassette in the vector facilitates the negative selection of cells in which random chromosomal insertion of the vector takes place. LoxP sequences allow the Cre-mediated deletion of the NeoR coding sequence. The SV40pA sequence helps to ensure disruption of gene expression after deletion of the NeoR sequence. (B) Anti-HOIP and anti-FLAG Western blots of cell lysates from A20.2J cells and HOIP-deficient (HOIP-/-) clones. A partial decrease in HOIP protein expression is evident in cells following disruption of one copy of Rnf31 (HOIP−/+). HOIP expression in clones reconstituted with an empty retroviral vector (pMIP) or a retroviral vector encoding FLAG-tagged HOIP is also shown. Approximate molecular weight of HOIP is 120 kD. To enable us to confirm that any signaling or functional defects observed in HOIP-deficient cells were due to disruption of the Rnf31 gene, both HOIP-deficient cell lines were transduced with a retrovirus encoding FLAG-tagged wild-type HOIP, thus restoring HOIP protein expression (Fig. 1B). These cells are referred to as HOIP-reconstituted cells. In the experiments that follow, responses by these cells were compared to those of A20.2J cells and HOIP-deficient cells transduced with a retroviral vector (pMIP) lacking a cDNA insert (empty vector). HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription Engagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31. 10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells. Cells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling. 10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells. A20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone. HOIP mediates CD40-stimulated NF-κB and JNK activation The results described above show that HOIP plays an important role in CD40-mediated effector functions of B cells. It follows that HOIP is likely a key mediator of CD40 signaling. To test this hypothesis, we stimulated A20.2J and HOIP-deficient cells with CD154 (CD40 ligand) expressed by HI5 insect cells [7], [8] and measured activation of the NF-κB and JNK pathways, two of the major transcriptional regulators activated by CD40 [4]. Cell-associated CD154 was used as the stimulus in these experiments as it typically provides more robust, and therefore more readily detected, activation signals than does anti-CD40 antibody. Activation of the canonical NF-κB pathway is initiated with the phosphorylation of IκB proteins by the IκB kinase complex (IKK). In resting cells, IκB proteins are responsible for sequestering NF-κB subunits in the cytoplasm. Phosphorylation by the IKK complex
T2636 4680-4683 Protein denotes IKK
T2637 4698-4702 Protein denotes CD40
T2642 4884-4899 Protein denotes CD40 engagement
T3954 4961-4971 Transcription denotes disruption
T3953 4975-4980 Protein denotes Rnf31
T3955 5074-5078 Protein denotes CD40
T3957 5202-5213 Binding denotes interaction
T3956 5217-5221 Protein denotes CD40
T3964 5326-5337 Binding denotes recruitment
T3958 5341-5345 Protein denotes HOIP
T3959 5349-5353 Protein denotes CD40
T3960 5358-5363 Protein denotes TRAF2
T3965 5383-5397 Gene_expression denotes overexpression
T3966 5383-5397 Positive_regulation denotes overexpression
T3961 5413-5424 Protein denotes HOIP mutant
T3962 5445-5449 Protein denotes CD40
T3963 5459-5464 Protein denotes NF-κB
T3967 5465-5475 Positive_regulation denotes activation
T3968 5540-5544 Protein denotes CD40
T3969 5632-5636 Protein denotes CD40
T3970 5822-5826 Protein denotes CD40
T3971 6033-6038 Protein denotes Rnf31
T3972 6298-6303 Protein denotes Rnf31
T3973 6377-6382 Protein denotes Rnf31
T3974 6471-6486 Protein denotes Cre recombinase
T3975 6609-6614 Protein denotes Rnf31
T3976 6699-6711 Protein denotes HOIP protein
T3977 6712-6722 Gene_expression denotes expression
T3978 6727-6739 Gene_expression denotes undetectable
T15300 7063-7068 Protein denotes Rnf31
T15301 7104-7113 Protein denotes HOIP gene
T15302 7345-7350 Protein denotes Rnf31
T15305 7409-7418 Localization denotes insertion
T15306 7409-7418 Localization denotes insertion
T15303 7422-7449 Protein denotes neomycin phosphotransferase
T15304 7451-7455 Protein denotes NeoR
T15310 7674-7679 Negative_regulation denotes allow
T15307 7684-7687 Protein denotes Cre
T15311 7688-7696 Positive_regulation denotes mediated
T15309 7697-7705 Negative_regulation denotes deletion
T15308 7713-7733 Protein denotes NeoR coding sequence
T15313 7807-7815 Negative_regulation denotes deletion
T15312 7823-7836 Protein denotes NeoR sequence
T15317 7961-7969 Negative_regulation denotes decrease
T15314 7973-7985 Protein denotes HOIP protein
T15316 7986-7996 Gene_expression denotes expression
T15315 8053-8058 Protein denotes Rnf31
T15318 8193-8197 Protein denotes HOIP
T3981 8373-8383 Negative_regulation denotes disruption
T3979 8391-8401 Protein denotes Rnf31 gene
T3980 8520-8532 Protein denotes HOIP protein
T3982 8533-8543 Gene_expression denotes expression
T5267 8823-8831 Positive_regulation denotes required
T5261 8836-8840 Protein denotes CD40
T5262 8850-8854 Protein denotes CD80
T5264 8855-8867 Positive_regulation denotes upregulation
T5266 8872-8882 Positive_regulation denotes activation
T5263 8886-8902 Protein denotes germline epsilon
T5265 8903-8916 Transcription denotes transcription
T5270 8917-8927 Binding denotes Engagement
T5268 8931-8935 Protein denotes CD40
T5272 8947-8958 Positive_regulation denotes upregulates
T5271 8959-8969 Gene_expression denotes expression
T5269 8973-8977 Protein denotes CD80
T5273 9138-9142 Protein denotes CD40
T5276 9169-9181 Positive_regulation denotes upregulation
T5274 9185-9189 Protein denotes CD80
T5277 9201-9211 Binding denotes engagement
T5275 9215-9219 Protein denotes CD40
T5278 9335-9339 Protein denotes CD40
T5280 9351-9363 Positive_regulation denotes upregulation
T5279 9367-9371 Protein denotes CD80
T5281 9389-9396 Negative_regulation denotes reduced
T5285 9446-9458 Positive_regulation denotes upregulation
T5282 9462-9466 Protein denotes CD80
T5288 9529-9535 Negative_regulation denotes defect
T5283 9539-9543 Protein denotes CD80
T5286 9544-9556 Positive_regulation denotes upregulation
T5287 9600-9610 Negative_regulation denotes disruption
T5284 9614-9619 Protein denotes Rnf31
T15644 9665-9669 Protein denotes CD40
T15648 9670-9678 Positive_regulation denotes mediated
T15645 9679-9683 Protein denotes CD80
T15646 9684-9696 Positive_regulation denotes upregulation
T15647 9700-9709 Negative_regulation denotes defective
T15649 9775-9799 Protein denotes isotype control antibody
T15650 9815-9819 Protein denotes CD40
T15654 9858-9868 Gene_expression denotes expression
T15651 9872-9876 Protein denotes CD80
T15652 9922-9926 Protein denotes CD80
T15653 9972-9996 Protein denotes isotype control antibody
T15657 10089-10097 Negative_regulation denotes reversed
T15655 10108-10112 Protein denotes CD80
T15656 10113-10125 Positive_regulation denotes upregulation
T5289 10239-10243 Protein denotes CD40
T5290 10329-10343 Protein denotes immunoglobulin
T5291 10397-10411 Protein denotes immunoglobulin
T5296 10478-10488 Gene_expression denotes expressing
T5292 10489-10492 Protein denotes IgM
T5297 10497-10503 Binding denotes switch
T5293 10507-10510 Protein denotes IgA
T5294 10512-10515 Protein denotes IgG
T5295 10520-10523 Protein denotes IgE
T5298 10623-10626 Protein denotes IgE
T5302 10634-10641 Positive_regulation denotes induced
T5301 10649-10660 Binding denotes combination
T5299 10664-10668 Protein denotes IL-4
T5300 10673-10685 Protein denotes CD40 signals
T5306 10710-10719 Positive_regulation denotes necessary
T5303 10724-10727 Protein denotes IgE
T5305 10728-10738 Gene_expression denotes production
T5304 10830-10833 Protein denotes IgE
T5307 10996-11000 Protein denotes CD40
T5308 11020-11024 Protein denotes IL-4
T5309 11099-11117 Protein denotes Anti-CD40 antibody
T5311 11118-11129 Positive_regulation denotes stimulation
T5310 11153-11157 Protein denotes IL-4
T5312 11335-11339 Protein denotes CD40
T5313 11363-11367 Protein denotes CD40
T5314 11372-11376 Protein denotes IL-4
T5315 11467-11485 Protein denotes anti-CD40 antibody
T5316 11607-11611 Protein denotes CD40
T16092 11701-11705 Protein denotes CD40
T16093 11992-11996 Protein denotes CD40
T16094 12003-12027 Protein denotes isotype control antibody
T16095 12029-12032 Protein denotes iso
T16096 12051-12055 Protein denotes IL-4
T16097 12173-12188 Protein denotes GLε transcripts
T16098 12231-12248 Protein denotes Hprt1 transcripts
T6224 12491-12499 Positive_regulation denotes mediates
T6220 12500-12504 Protein denotes CD40
T6225 12505-12515 Positive_regulation denotes stimulated
T6221 12516-12521 Protein denotes NF-κB
T6222 12526-12529 Protein denotes JNK
T6223 12530-12540 Positive_regulation denotes activation
T6226 12530-12540 Positive_regulation denotes activation
T6227 12611-12615 Protein denotes CD40
T6228 12705-12709 Protein denotes CD40
T6229 12797-12802 Protein denotes CD154
T6230 12804-12808 Protein denotes CD40
T6233 12817-12826 Gene_expression denotes expressed
T6234 12817-12826 Gene_expression denotes expressed
T6235 12869-12879 Positive_regulation denotes activation
T6236 12869-12879 Positive_regulation denotes activation
T6231 12887-12892 Protein denotes NF-κB
T6232 12897-12900 Protein denotes JNK
T6237 12994-12999 Protein denotes CD154
T6238 13155-13159 Protein denotes CD40
T6243 13170-13180 Positive_regulation denotes Activation
T6239 13198-13203 Protein denotes NF-κB
T6244 13234-13249 Phosphorylation denotes phosphorylation
T6245 13234-13249 Phosphorylation denotes phosphorylation
T6246 13234-13249 Phosphorylation denotes phosphorylation
T6240 13253-13265 Protein denotes IκB proteins
T6241 13273-13291 Protein denotes IκB kinase complex
T6242 13293-13296 Protein denotes IKK
T6247 13317-13329 Protein denotes IκB proteins
T6249 13350-13362 Positive_regulation denotes sequestering
T6248 13363-13377 Protein denotes NF-κB subunits
T6253 13396-13411 Phosphorylation denotes Phosphorylation
T6250 13419-13422 Protein denotes IKK
T6254 13431-13438 Regulation denotes targets
T6257 13431-13438 Regulation denotes targets
T6251 13439-13442 Protein denotes IκB
T6255 13475-13486 Protein_catabolism denotes degradation
T6252 13497-13502 Protein denotes NF-κB
T6256 13516-13523 Entity denotes nucleus
T6258 13554-13558 Protein denotes CD40
T6264 13559-13567 Positive_regulation denotes mediated
T6265 13559-13567 Positive_regulation denotes mediated
T6260 13568-13583 Phosphorylation denotes phosphorylation
T6261 13588-13599 Protein_catabolism denotes degradation
T6259 13603-13607 Protein denotes IκBα
T6262 13649-13657 Negative_regulation denotes impaired
T6263 13649-13657 Negative_regulation denotes impaired
T6269 13735-13745 Positive_regulation denotes activation
T6270 13735-13745 Positive_regulation denotes activation
T6266 13753-13784 Protein denotes stress-activated protein kinase
T6267 13785-13788 Protein denotes JNK
T6268 13804-13808 Protein denotes CD40
T6271 13809-13819 Binding denotes engagement
T6272 13830-13834 Protein denotes CD40
T6277 13835-13843 Positive_regulation denotes mediated
T6273 13844-13847 Protein denotes JNK
T6275 13848-13858 Positive_regulation denotes activation
T6278 13848-13858 Positive_regulation denotes activation
T6276 13911-13926 Phosphorylation denotes phosphorylation
T6274 13951-13954 Protein denotes JNK
T6279 13956-13960 Protein denotes CD40
T6280 13983-13988 Protein denotes NF-κB
T6281 13993-13996 Protein denotes JNK
T6284 14117-14124 Negative_regulation denotes absence
T6282 14128-14132 Protein denotes HOIP
T6283 14133-14143 Gene_expression denotes expression
T16631 14189-14193 Protein denotes CD40
T16635 14194-14201 Positive_regulation denotes induced
T16634 14202-14212 Positive_regulation denotes activation
T16636 14202-14212 Positive_regulation denotes activation
T16632 14216-14221 Protein denotes NF-κB
T16633 14226-14229 Protein denotes JNK
T16637 14268-14272 Protein denotes CD40
T16638 14558-14563 Protein denotes CD154
T16639 14564-14574 Gene_expression denotes expressing
T16641 14676-14683 Negative_regulation denotes lacking
T16640 14684-14689 Protein denotes CD154
T16642 14797-14809 Protein denotes Phospho-IκBα
T16643 14811-14816 Protein denotes pIκBα
T16644 14830-14833 Protein denotes JNK
T16645 14835-14839 Protein denotes pJNK
T16646 14884-14888 Protein denotes IκBα
T16647 14899-14902 Protein denotes JNK
T16648 14923-14926 Protein denotes JNK
T16651 14938-14947 Binding denotes recognize
T16652 14938-14947 Binding denotes recognize
T16649 14948-14951 Protein denotes p46
T16650 14956-14968 Protein denotes p54 isoforms
T16653 14971-14981 Protein denotes IκBα blots
T16654 15005-15010 Protein denotes actin
T8616 15297-15301 Protein denotes CD40
T8617 15446-15450 Protein denotes CD40
T8621 15529-15538 Binding denotes recruited
T8618 15546-15550 Protein denotes CD40
T8619 15574-15579 Protein denotes TRAF2
T8620 15643-15648 Protein denotes TRAF2
T8622 15689-15694 Protein denotes TRAF2
T8623 15715-15720 Protein denotes cIAP1
T8624 15725-15730 Protein denotes cIAP2
T8627 15750-15761 Binding denotes recruitment
T8625 15765-15769 Protein denotes HOIP
T8626 15773-15778 Protein denotes TNFR1
T8630 15831-15842 Binding denotes association
T8628 15856-15860 Protein denotes CD40
T8631 15919-15928 Negative_regulation denotes inhibitor
T8629 15932-15936 Protein denotes cIAP
T8632 16069-16081 Protein denotes SMAC peptide
T8637 16095-16102 Negative_regulation denotes reduced
T8633 16117-16122 Protein denotes cIAP1
T8636 16123-16133 Binding denotes associated
T8634 16143-16147 Protein denotes CD40
T8635 16196-16200 Protein denotes CD40
T8642 16300-16308 Negative_regulation denotes decrease
T8638 16359-16363 Protein denotes CD40
T8640 16364-16383 Binding denotes immunoprecipitation
T8641 16452-16462 Binding denotes recognized
T8639 16466-16484 Protein denotes anti-HOIP antibody
T8643 16518-16530 Protein denotes SMAC peptide
T8644 16699-16703 Protein denotes CD40
T8645 16824-16837 Protein denotes cIAP proteins
T8646 16909-16913 Protein denotes CD40
T17868 16975-16978 Protein denotes IKK
T17870 16979-16990 Binding denotes recruitment
T17869 16998-17002 Protein denotes CD40
T17874 17086-17093 Negative_regulation denotes reduces
T17873 17094-17105 Binding denotes recruitment
T17871 17109-17114 Protein denotes cIAP1
T17872 17118-17122 Protein denotes CD40
T17875 17500-17509 Protein denotes anti-CD40
T17876 17516-17540 Protein denotes isotype control antibody
T17877 17749-17754 Protein denotes cIAP1
T17878 17756-17761 Protein denotes TRAF2
T17879 17763-17768 Protein denotes TRAF3
T17880 17895-17899 Protein denotes CD40
T17888 18088-18106 Binding denotes immunoprecipitated
T17881 18153-18171 Protein denotes anti-CD40 antibody
T17882 18209-18213 Protein denotes CD40
T17883 18215-18220 Protein denotes TRAF2
T17884 18222-18227 Protein denotes TRAF3
T17885 18229-18234 Protein denotes cIAP1
T17886 18236-18240 Protein denotes HOIP
T17887 18242-18248 Protein denotes IKKα/β
T17892 18295-18303 Positive_regulation denotes required
T17891 18308-18323 Binding denotes coprecipitation
T17889 18327-18339 Protein denotes IKK proteins
T17890 18345-18349 Protein denotes CD40
T17893 18554-18588 Protein denotes HOIP-dependent recruitment of IKKγ
T17894 18592-18596 Protein denotes CD40
T17895 18753-18757 Protein denotes IKKγ
T17896 18791-18795 Protein denotes IKKγ
T17897 18989-18995 Protein denotes BP tag
T17898 18997-19001 Protein denotes IKKγ
T17899 19020-19025 Protein denotes TRAF2
T17900 19031-19043 Protein denotes anti-CD40 IP
T17901 19079-19092 Protein denotes λ phosphatase
T17902 19285-19290 Protein denotes TRAF3
T8647 19493-19497 Protein denotes CD40
T8648 19521-19525 Protein denotes CD40
T8649 19631-19635 Protein denotes CD40
T8650 19719-19724 Protein denotes TRAF2
T8651 19726-19731 Protein denotes TRAF3
T8652 19733-19739 Protein denotes IKKα/β
T8653 19770-19775 Protein denotes TRAF2
T8654 19777-19782 Protein denotes TRAF3
T8655 19788-19793 Protein denotes cIAP1
T8656 19797-19801 Protein denotes CD40
T8658 19942-19953 Binding denotes association
T8659 19942-19953 Binding denotes association
T8660 19942-19953 Binding denotes association
T8657 19977-19981 Protein denotes CD40
T8661 19996-20011 Protein denotes IKKα/β and IKKγ
T8663 20021-20031 Gene_expression denotes detectable
T8664 20021-20031 Gene_expression denotes detectable
T8662 20035-20039 Protein denotes CD40
T8665 20123-20146 Protein denotes IKKα/β and IKKγ in CD40
T8666 20302-20305 Protein denotes IKK
T8668 20306-20317 Binding denotes recruitment
T8670 20359-20366 Negative_regulation denotes absence
T8667 20370-20374 Protein denotes HOIP
T8669 20375-20385 Gene_expression denotes expression
T8673 20433-20444 Binding denotes recruitment
T8671 20452-20457 Protein denotes NF-κB
T8672 20484-20488 Protein denotes CD40
T8674 20553-20557 Protein denotes IKKγ
T8675 20569-20573 Protein denotes CD40
T8676 20732-20736 Protein denotes IKKγ
T8677 20860-20870 Protein denotes mouse IKKγ
T8678 20889-20893 Protein denotes CD40
T8682 21246-21257 Binding denotes recruitment
T8679 21261-21265 Protein denotes IKKγ
T8680 21269-21273 Protein denotes CD40
T8683 21274-21282 Positive_regulation denotes requires
T8681 21283-21287 Protein denotes HOIP
T8684 21369-21373 Protein denotes CD40
T8685 21434-21452 Protein denotes lambda phosphatase
T8688 21559-21566 Negative_regulation denotes reduced
T8686 21575-21579 Protein denotes CD40
T8687 21591-21595 Protein denotes IKKγ
T8689 21808-21812 Protein denotes CD40
T8691 21813-21823 Binding denotes associated
T8690 21824-21828 Protein denotes IKKγ
T8692 21890-21905 Phosphorylation denotes phosphorylation
T8695 21969-21980 Binding denotes association
T8693 21984-21988 Protein denotes IKKγ
T8694 21994-21998 Protein denotes CD40
T11648 22155-22159 Protein denotes CD40
T11649 22268-22272 Protein denotes CD40
T11653 22282-22294 Positive_regulation denotes upregulation
T11650 22298-22302 Protein denotes CD80
T11652 22303-22313 Gene_expression denotes expression
T11651 22365-22397 Protein denotes immunoglobulin heavy chain locus
T11654 22562-22567 Protein denotes TRAF2
T11655 22575-22579 Protein denotes CD40
T11659 22630-22639 Positive_regulation denotes promoting
T11658 22644-22655 Binding denotes recruitment
T11656 22663-22674 Protein denotes IKK complex
T11657 22678-22682 Protein denotes CD40
T11660 22706-22710 Protein denotes CD40
T11664 22711-22718 Positive_regulation denotes induced
T11662 22719-22729 Positive_regulation denotes activation
T11665 22719-22729 Positive_regulation denotes activation
T11661 22733-22738 Protein denotes NF-κB
T11663 22742-22751 Positive_regulation denotes dependent
T11666 22809-22825 Protein denotes HOIP facilitates
T11669 22830-22840 Positive_regulation denotes activation
T11667 22844-22847 Protein denotes JNK
T11668 22863-22878 Protein denotes CD40 engagement
T11670 22974-22978 Protein denotes CD40
T11671 23022-23026 Protein denotes CD40
T11672 23204-23208 Protein denotes HOIP
T11675 23484-23492 Binding denotes interact
T11673 23498-23502 Protein denotes IKKγ
T11676 23518-23528 Positive_regulation denotes activation
T11674 23532-23537 Protein denotes NF-κB
T11680 23652-23660 Positive_regulation denotes mediates
T11679 23661-23672 Binding denotes recruitment
T11677 23676-23680 Protein denotes IKKγ
T11678 23688-23692 Protein denotes CD40
T11681 23725-23729 Protein denotes CD40
T11682 23873-23877 Protein denotes IKKγ
T11683 23893-23897 Protein denotes CD40
T11685 23898-23916 Binding denotes immunoprecipitates
T11686 24078-24085 Positive_regulation denotes enhance
T11684 24086-24090 Protein denotes IKKγ
T11692 24155-24163 Positive_regulation denotes mediates
T11690 24164-24175 Binding denotes recruitment
T11687 24183-24194 Protein denotes IKK complex
T11688 24198-24202 Protein denotes CD40
T11691 24224-24233 Binding denotes recruited
T11689 24237-24241 Protein denotes CD40
T11693 24290-24294 Protein denotes HOIP
T11694 24332-24335 Protein denotes IKK
T11697 24401-24412 Binding denotes recruitment
T11695 24420-24431 Protein denotes IKK complex
T11696 24435-24439 Protein denotes CD40
T11698 24454-24470 Protein denotes ubiquitin ligase
T11699 24548-24552 Protein denotes IKKγ
T11703 24572-24579 Binding denotes binding
T11700 24692-24696 Protein denotes CD40
T11704 24753-24760 Binding denotes recruit
T11701 24761-24765 Protein denotes IKKγ
T11702 24773-24777 Protein denotes CD40
T11705 24788-24795 Binding denotes complex
T11707 24838-24849 Binding denotes recruitment
T11706 24872-24876 Protein denotes CD40
T11710 24964-24975 Binding denotes recruitment
T11708 24979-24983 Protein denotes HOIP
T11709 25012-25017 Protein denotes TRAF2
T11711 25046-25051 Protein denotes TRAF2
T11712 25056-25078 Protein denotes HOIP directly interact
T11713 25108-25124 Protein denotes ubiquitin ligase
T11714 25152-25157 Protein denotes TRAF2
T11717 25203-25212 Binding denotes generates
T11715 25224-25244 Protein denotes polyubiquitin chains
T11718 25263-25267 Binding denotes bind
T11719 25277-25286 Binding denotes associate
T11720 25277-25286 Binding denotes associate
T11716 25296-25300 Protein denotes CD40
T11726 25383-25387 Binding denotes link
T11721 25396-25401 Protein denotes TRAF2
T11722 25406-25410 Protein denotes HOIP
T11723 25414-25418 Protein denotes CD40
T11727 25472-25481 Positive_regulation denotes dependent
T11728 25472-25481 Positive_regulation denotes dependent
T11724 25487-25492 Protein denotes TRAF6
T11725 25504-25509 Protein denotes TRAF2
T11729 25540-25544 Protein denotes TRAF
T11737 25545-25554 Negative_regulation denotes deficient
T11738 25587-25597 Positive_regulation denotes activation
T11730 25601-25606 Protein denotes NF-κB
T11731 25610-25614 Protein denotes CD40
T11739 25624-25632 Positive_regulation denotes mediated
T11740 25624-25632 Positive_regulation denotes mediated
T11732 25643-25648 Protein denotes TRAF2
T11733 25652-25657 Protein denotes TRAF6
T11741 25665-25675 Positive_regulation denotes activation
T11734 25679-25682 Protein denotes JNK
T11735 25686-25690 Protein denotes CD40
T11742 25703-25712 Positive_regulation denotes dependent
T11736 25716-25721 Protein denotes TRAF6
T11743 25765-25769 Protein denotes CD40
T11750 25770-25778 Positive_regulation denotes mediated
T11751 25770-25778 Positive_regulation denotes mediated
T11748 25779-25789 Positive_regulation denotes activation
T11749 25779-25789 Positive_regulation denotes activation
T11744 25798-25803 Protein denotes NF-κB
T11745 25808-25811 Protein denotes JNK
T11746 25854-25859 Protein denotes TRAF2
T11747 25864-25869 Protein denotes TRAF6
T11752 25941-25945 Protein denotes CD40
T11759 25955-25965 Positive_regulation denotes activation
T11760 25955-25965 Positive_regulation denotes activation
T11753 25969-25974 Protein denotes NF-κB
T11754 25979-25982 Protein denotes JNK
T11755 25990-25995 Protein denotes TRAF6
T11756 26069-26074 Protein denotes TRAF6
T11757 26104-26108 Protein denotes CD40
T11758 26175-26180 Protein denotes TRAF6
T11761 26219-26224 Protein denotes TRAF6
T11763 26243-26247 Binding denotes bind
T11762 26248-26252 Protein denotes CD40
T11764 26535-26539 Protein denotes CD40
T11765 26673-26678 Protein denotes TRAF2
T11766 26682-26687 Protein denotes TRAF6
T11769 26718-26728 Binding denotes associated
T11767 26757-26781 Protein denotes TNF receptor superfamily
T11768 26790-26809 Protein denotes Toll-like receptors
T11770 26839-26843 Protein denotes HOIP
T11771 26867-26878 Protein denotes TNFR family
T11775 26944-26953 Binding denotes interacts
T11776 26944-26953 Binding denotes interacts
T11772 26978-26985 Protein denotes SHARPIN
T11773 26987-26992 Protein denotes SHANK
T11774 27004-27006 Protein denotes RH
T11777 27256-27263 Protein denotes SHARPIN
T11779 27444-27454 Gene_expression denotes production
T11778 27467-27481 Protein denotes immunoglobulin
T11780 27551-27558 Protein denotes SHARPIN
T11781 27563-27567 Protein denotes HOIP
T11783 27653-27663 Regulation denotes regulation
T11782 27679-27683 Protein denotes HOIP
T12051 27909-27919 Gene_expression denotes expressing
T12050 27920-27925 Protein denotes CD154
T12290 27964-27998 Protein denotes Mouse anti-birch profilin antibody
T12291 28026-28030 Protein denotes CD40
T12292 28215-28230 Protein denotes anti-mouse CD80
T12293 28411-28428 Protein denotes Goat anti-rat IgG
T12294 28434-28437 Protein denotes HRP
T12859 28739-28744 Protein denotes Rnf31
T12860 29711-29715 Protein denotes NeoR
T12861 29746-29751 Protein denotes Rnf31
T12864 29808-29818 Gene_expression denotes expression
T12862 29836-29839 Protein denotes Cre
T12863 29860-29864 Protein denotes NeoR
T12865 29970-29975 Protein denotes Rnf31
T12866 30043-30046 Protein denotes PCR
T12867 30051-30063 Protein denotes HOIP protein
T12868 30064-30074 Gene_expression denotes expression
T12869 30064-30074 Gene_expression denotes expression
T13321 30195-30224 Protein denotes HOIP and IKKγ cDNA constructs
T13323 30250-30259 Negative_regulation denotes replacing
T13322 30273-30276 Protein denotes GFP
T13324 30393-30408 Protein denotes mouse HOIP cDNA
T13325 30462-30478 Protein denotes A pMIP construct
T13326 30488-30498 Protein denotes mouse IKKγ
T13327 30550-30553 Protein denotes tag
T13328 30674-30683 Protein denotes pMIP-IKKγ
T13519 31138-31142 Protein denotes CD80
T13520 31143-31155 Positive_regulation denotes upregulation
T13521 31258-31262 Protein denotes CD40
T13522 31269-31293 Protein denotes isotype control antibody
T13838 31569-31573 Protein denotes CD40
T13839 31716-31720 Protein denotes CD40
T13840 31959-31964 Protein denotes Hprt1
T13842 32148-32158 Gene_expression denotes expression
T13841 32162-32167 Protein denotes Hprt1
T14258 32170-32175 Protein denotes NF-κB
T14259 32180-32183 Protein denotes JNK
T14260 32184-32194 Positive_regulation denotes activation
T14261 32220-32224 Protein denotes CD40
T14264 32279-32289 Gene_expression denotes expressing
T14265 32279-32289 Gene_expression denotes expressing
T14262 32296-32301 Protein denotes CD154
T14263 32303-32307 Protein denotes CD40
T14266 32492-32495 Protein denotes Fig
T14267 32862-32865 Protein denotes Fig
T14732 33136-33140 Protein denotes CD40
T14733 33382-33394 Protein denotes anti-rat IgG
T14734 33396-33403 Protein denotes Jackson
T14735 33421-33425 Protein denotes CD40
T14736 33427-33431 Protein denotes 1C10
T14738 33693-33709 Phosphorylation denotes dephosphorylated
T14737 33715-33733 Protein denotes lambda phosphatase
R10088 T12862 T12864 themeOf Cre,expression
R10089 T12866 T12868 themeOf PCR,expression
R10090 T12867 T12869 themeOf HOIP protein,expression
R10475 T13322 T13323 themeOf GFP,replacing
R10634 T13519 T13520 themeOf CD80,upregulation
R10887 T13841 T13842 themeOf Hprt1,expression
R11252 T14258 T14260 themeOf NF-κB,activation
R11253 T14262 T14264 themeOf CD154,expressing
R11254 T14263 T14265 themeOf CD40,expressing
R11682 T14737 T14738 themeOf lambda phosphatase,dephosphorylated
R12133 T15303 T15305 themeOf neomycin phosphotransferase,insertion
R12134 T15304 T15306 themeOf NeoR,insertion
R12135 T15307 T15311 causeOf Cre,mediated
R12136 T15308 T15309 themeOf NeoR coding sequence,deletion
R12137 T15309 T15310 themeOf deletion,allow
R12138 T15309 T15311 themeOf deletion,mediated
R12139 T15312 T15313 themeOf NeoR sequence,deletion
R12140 T15314 T15316 themeOf HOIP protein,expression
R12141 T15316 T15317 themeOf expression,decrease
R12390 T15644 T15648 causeOf CD40,mediated
R12391 T15645 T15646 themeOf CD80,upregulation
R12392 T15646 T15647 themeOf upregulation,defective
R12393 T15646 T15648 themeOf upregulation,mediated
R12394 T15651 T15654 themeOf CD80,expression
R12395 T15655 T15656 themeOf CD80,upregulation
R12396 T15656 T15657 themeOf upregulation,reversed
R13124 T16631 T16635 causeOf CD40,induced
R13125 T16632 T16634 themeOf NF-κB,activation
R13126 T16634 T16635 themeOf activation,induced
R13127 T16635 T16636 themeOf induced,activation
R13128 T16638 T16639 themeOf CD154,expressing
R13129 T16640 T16641 themeOf CD154,lacking
R13130 T16649 T16651 themeOf p46,recognize
R13131 T16650 T16652 themeOf p54 isoforms,recognize
R14086 T17868 T17870 themeOf IKK,recruitment
R14087 T17869 T17870 themeOf CD40,recruitment
R14088 T17871 T17873 themeOf cIAP1,recruitment
R14089 T17872 T17873 themeOf CD40,recruitment
R14090 T17873 T17874 themeOf recruitment,reduces
R14091 T17881 T17888 themeOf anti-CD40 antibody,immunoprecipitated
R14092 T17889 T17891 themeOf IKK proteins,coprecipitation
R14093 T17890 T17891 themeOf CD40,coprecipitation
R14094 T17891 T17892 themeOf coprecipitation,required
R1955 T2574 T2582 themeOf TNF receptor-associated factor,important
R1957 T2574 T2584 themeOf TNF receptor-associated factor,important
R1958 T2574 T2586 themeOf TNF receptor-associated factor,important
R1959 T2574 T2588 themeOf TNF receptor-associated factor,important
R1960 T2574 T2581 partOf TNF receptor-associated factor,family
R1961 T2575 T2583 themeOf TRAF,important
R1962 T2575 T2585 themeOf TRAF,important
R1963 T2575 T2587 themeOf TRAF,important
R1964 T2575 T2589 themeOf TRAF,important
R1965 T2575 T2581 partOf TRAF,family
R1966 T2562 T2565 themeOf CD40,activated
R1967 T2576 T2582 causeOf TRAF1,important
R1968 T2563 T2566 themeOf CD40,expressed
R1969 T2564 T2567 themeOf CD154,expressed
R1970 T2568 T2571 themeOf CD40,engagement
R1971 T2576 T2583 causeOf TRAF1,important
R1972 T2577 T2584 causeOf TRAF2,important
R1973 T2577 T2585 causeOf TRAF2,important
R1974 T2578 T2586 causeOf TRAF3,important
R1975 T2578 T2587 causeOf TRAF3,important
R1976 T2579 T2588 causeOf TRAF6,important
R1977 T2613 T2614 themeOf NF-κB,activation
R1978 T2579 T2589 causeOf TRAF6,important
R1979 T2614 T2615 themeOf activation,necessary
R1980 T2581 T2582 Site family,important
R1981 T2581 T2583 Site family,important
R1982 T2619 T2623 themeOf CD40,production
R1983 T2620 T2624 themeOf IL-4,production
R1984 T2581 T2584 Site family,important
R1985 T2621 T2625 themeOf immunoglobulin epsilon heavy chain locus,production
R1986 T2581 T2585 Site family,important
R1987 T2581 T2586 Site family,important
R1988 T2581 T2587 Site family,important
R1989 T2581 T2588 Site family,important
R1990 T2581 T2589 Site family,important
R1991 T2626 T2630 causeOf CD40,activation
R1992 T2626 T2633 causeOf CD40,activation
R1993 T2627 T2630 themeOf NF-κB,activation
R1994 T2590 T2591 themeOf CD40,binding
R1995 T2630 T2631 themeOf activation,stress-activated
R1996 T2630 T2632 themeOf activation,defective
R1997 T2631 T2633 themeOf stress-activated,activation
R1998 T2634 T2638 themeOf NF-κB,activation
R1999 T2635 T2640 themeOf IκB kinase,association
R2000 T2636 T2641 themeOf IKK,association
R2001 T2637 T2639 themeOf CD40,association
R2002 T2637 T2640 themeOf CD40,association
R2003 T2637 T2641 themeOf CD40,association
R2004 T2595 T2600 themeOf TRAFs,associate
R2005 T2596 T2600 themeOf CD40,associate
R2006 T2601 T2607 themeOf HOIL-1L interacting protein,recruited
R2007 T2602 T2608 themeOf HOIP,recruited
R2008 T2603 T2609 themeOf ubiquitin ligase,recruited
R2009 T2604 T2607 themeOf CD40,recruited
R2011 T2604 T2608 themeOf CD40,recruited
R2013 T2604 T2609 themeOf CD40,recruited
R2014 T2606 T2610 themeOf CD40,engagement
R3082 T3953 T3954 themeOf Rnf31,disruption
R3087 T3956 T3957 themeOf CD40,interaction
R3088 T3958 T3964 themeOf HOIP,recruitment
R3089 T3959 T3964 themeOf CD40,recruitment
R3090 T3961 T3965 themeOf HOIP mutant,overexpression
R3091 T3963 T3967 themeOf NF-κB,activation
R3092 T3965 T3966 themeOf overexpression,overexpression
R3093 T3976 T3977 themeOf HOIP protein,expression
R3094 T3979 T3981 themeOf Rnf31 gene,disruption
R3095 T3980 T3982 themeOf HOIP protein,expression
R3096 T3976 T3978 themeOf HOIP protein,undetectable
R4084 T5262 T5264 themeOf CD80,upregulation
R4085 T5263 T5265 themeOf germline epsilon,transcription
R4086 T5265 T5266 themeOf transcription,activation
R4087 T5266 T5267 themeOf activation,required
R4088 T5268 T5270 themeOf CD40,Engagement
R4089 T5269 T5271 themeOf CD80,expression
R4090 T5270 T5272 causeOf Engagement,upregulates
R4091 T5271 T5272 themeOf expression,upregulates
R4092 T5274 T5276 themeOf CD80,upregulation
R4093 T5275 T5277 themeOf CD40,engagement
R4094 T5278 T5280 causeOf CD40,upregulation
R4095 T5279 T5280 themeOf CD80,upregulation
R4096 T5280 T5281 themeOf upregulation,reduced
R4097 T5282 T5285 themeOf CD80,upregulation
R4098 T5283 T5286 themeOf CD80,upregulation
R4099 T5284 T5287 themeOf Rnf31,disruption
R4100 T5286 T5288 themeOf upregulation,defect
R4101 T5292 T5296 themeOf IgM,expressing
R4102 T5295 T5297 themeOf IgE,switch
R4103 T5298 T5302 themeOf IgE,induced
R4104 T5300 T5301 themeOf CD40 signals,combination
R4105 T5301 T5302 causeOf combination,induced
R4106 T5303 T5305 themeOf IgE,production
R4107 T5305 T5306 themeOf production,necessary
R4108 T5309 T5311 themeOf Anti-CD40 antibody,stimulation
R4785 T6220 T6225 causeOf CD40,stimulated
R4786 T6221 T6223 themeOf NF-κB,activation
R4787 T6223 T6224 themeOf activation,mediates
R4788 T6223 T6225 themeOf activation,stimulated
R4789 T6225 T6226 themeOf stimulated,activation
R4790 T6229 T6233 themeOf CD154,expressed
R4791 T6230 T6234 themeOf CD40,expressed
R4792 T6231 T6235 themeOf NF-κB,activation
R4793 T6232 T6236 themeOf JNK,activation
R4794 T6239 T6243 themeOf NF-κB,Activation
R4795 T6240 T6244 themeOf IκB proteins,phosphorylation
R4796 T6241 T6245 themeOf IκB kinase complex,phosphorylation
R4797 T6242 T6246 themeOf IKK,phosphorylation
R4798 T6247 T6249 causeOf IκB proteins,sequestering
R4799 T6248 T6249 themeOf NF-κB subunits,sequestering
R4800 T6250 T6253 themeOf IKK,Phosphorylation
R4801 T6251 T6254 themeOf IκB,targets
R4802 T6251 T6255 themeOf IκB,degradation
R4803 T6253 T6254 causeOf Phosphorylation,targets
R4804 T6253 T6257 causeOf Phosphorylation,targets
R4805 T6255 T6257 themeOf degradation,targets
R4806 T6258 T6264 causeOf CD40,mediated
R4807 T6258 T6265 causeOf CD40,mediated
R4808 T6259 T6260 themeOf IκBα,phosphorylation
R4809 T6259 T6261 themeOf IκBα,degradation
R4810 T6260 T6262 themeOf phosphorylation,impaired
R4811 T6260 T6264 themeOf phosphorylation,mediated
R4812 T6261 T6263 themeOf degradation,impaired
R4813 T6261 T6265 themeOf degradation,mediated
R4814 T6266 T6269 themeOf stress-activated protein kinase,activation
R4815 T6267 T6270 themeOf JNK,activation
R4816 T6268 T6271 themeOf CD40,engagement
R4817 T6272 T6277 causeOf CD40,mediated
R4818 T6273 T6275 themeOf JNK,activation
R4819 T6274 T6276 themeOf JNK,phosphorylation
R4820 T6275 T6277 themeOf activation,mediated
R4821 T6277 T6278 themeOf mediated,activation
R4822 T6282 T6283 themeOf HOIP,expression
R4823 T6283 T6284 themeOf expression,absence
R653 T894 T896 themeOf CD40,engagement
R654 T898 T901 themeOf CD40,engagement
R655 T899 T902 themeOf NF-κB,activation
R656 T900 T903 themeOf stress-activated protein kinases,activation
R657 T910 T917 themeOf HOIL-1L-interacting protein,recruitment
R658 T911 T915 themeOf HOIP,recruitment
R659 T911 T918 themeOf HOIP,recruitment
R660 T912 T916 themeOf CD40,recruitment
R661 T912 T917 themeOf CD40,recruitment
R662 T912 T918 themeOf CD40,recruitment
R663 T912 T914 partOf CD40,domain
R664 T914 T915 Site domain,recruitment
R665 T914 T916 Site domain,recruitment
R666 T914 T917 Site domain,recruitment
R667 T914 T918 Site domain,recruitment
R668 T923 T924 themeOf germline immunoglobulin,transcription
R669 T924 T925 themeOf transcription,activation
R670 T926 T929 causeOf CD40,activation
R671 T926 T930 causeOf CD40,activation
R672 T927 T929 themeOf NF-κB,activation
R6724 T8625 T8627 themeOf HOIP,recruitment
R673 T928 T930 themeOf c-Jun kinase,activation
R674 T929 T931 themeOf activation,impaired
R6744 T8626 T8627 themeOf TNFR1,recruitment
R6745 T8628 T8630 themeOf CD40,association
R6746 T8629 T8631 themeOf cIAP,inhibitor
R6747 T8633 T8636 themeOf cIAP1,associated
R6748 T8613 T8614 themeOf "IκB kinase (IKK) complex with CD40 was undetectable in HOIP-deficient cells. Together, our results indicate that HOIP plays a critical role in the activation of signaling pathways that regulate cellular responses to CD40 engagement. Results Generation of HOIP-deficient B cells via targeted disruption of Rnf31, the gene encoding HOIP In our previous study, we demonstrated that HOIP is recruited to the CD40 signaling complex in two mouse B cell lines [6]. Similar results were obtained with mouse splenocytes, indicating that the interaction of CD40 with HOIP is not limited to transformed B cell lines (unpublished observations). We also found that the recruitment of HOIP to CD40 was TRAF2-dependent and that overexpression of a truncated HOIP mutant partially inhibited CD40-mediated NF-κB activation. These results support the hypothesis that HOIP plays a role in CD40 signal transduction. To further define the role and evaluate the importance of HOIP in CD40 signaling, we used somatic cell gene targeting to disrupt the gene encoding HOIP in the mouse B cell line A20.2J. This cell line has been particularly useful in the characterization of CD40 signaling mechanisms due to the relative ease with which its genome can be modified by homologous recombination [7], [8], [9]. We used a targeting vector capable of undergoing homologous recombination with Rnf31 (the gene encoding HOIP) to disrupt the coding sequence of the gene in exon 5 (Fig. 1A). Following introduction of the vector, the neomycin-resistant clones that arose were screened by PCR amplification of genomic DNA to identify cells containing a disrupted Rnf31 allele. To remove the selectable marker gene cassette from the disrupted Rnf31 allele, recombinant cell lines were transiently transfected with a plasmid that encodes Cre recombinase. This step allowed us to perform a second round of targeting and drug selection, generating cells in which both copies of Rnf31 were disrupted. Two independent clonal cell lines were chosen for further analysis. HOIP protein expression was undetectable in both cell lines, as determined by Western blot analysis of cell lysates (Fig. 1B), demonstrating that the targeting process was successful. In the text that follows, the two gene-targeted cell lines are referred to as HOIP-deficient cells. 10.1371/journal.pone.0023061.g001 Figure 1 HOIP gene targeting. (A) Regions of Rnf31 gene sequence (bottom) used in the HOIP gene targeting vector (top) are shown. Arrows (F and R) indicate approximate positions of sequences homologous to oligonucleotides used for PCR-mediated detection of homologous recombination. Homologous recombination of the vector with Rnf31 resulted in a small chromosomal deletion and the in-frame insertion of neomycin phosphotransferase (NeoR) into the amino-terminal HOIP coding sequence. A diphtheria toxin (DT) cassette in the vector facilitates the negative selection of cells in which random chromosomal insertion of the vector takes place. LoxP sequences allow the Cre-mediated deletion of the NeoR coding sequence. The SV40pA sequence helps to ensure disruption of gene expression after deletion of the NeoR sequence. (B) Anti-HOIP and anti-FLAG Western blots of cell lysates from A20.2J cells and HOIP-deficient (HOIP-/-) clones. A partial decrease in HOIP protein expression is evident in cells following disruption of one copy of Rnf31 (HOIP−/+). HOIP expression in clones reconstituted with an empty retroviral vector (pMIP) or a retroviral vector encoding FLAG-tagged HOIP is also shown. Approximate molecular weight of HOIP is 120 kD. To enable us to confirm that any signaling or functional defects observed in HOIP-deficient cells were due to disruption of the Rnf31 gene, both HOIP-deficient cell lines were transduced with a retrovirus encoding FLAG-tagged wild-type HOIP, thus restoring HOIP protein expression (Fig. 1B). These cells are referred to as HOIP-reconstituted cells. In the experiments that follow, responses by these cells were compared to those of A20.2J cells and HOIP-deficient cells transduced with a retroviral vector (pMIP) lacking a cDNA insert (empty vector). HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription Engagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31. 10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells. Cells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling. 10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells. A20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone. HOIP mediates CD40-stimulated NF-κB and JNK activation The results described above show that HOIP plays an important role in CD40-mediated effector functions of B cells. It follows that HOIP is likely a key mediator of CD40 signaling. To test this hypothesis, we stimulated A20.2J and HOIP-deficient cells with CD154 (CD40 ligand) expressed by HI5 insect cells [7], [8] and measured activation of the NF-κB and JNK pathways, two of the major transcriptional regulators activated by CD40 [4]. Cell-associated CD154 was used as the stimulus in these experiments as it typically provides more robust, and therefore more readily detected, activation signals than does anti-CD40 antibody. Activation of the canonical NF-κB pathway is initiated with the phosphorylation of IκB proteins by the IκB kinase complex (IKK). In resting cells, IκB proteins are responsible for sequestering NF-κB subunits in the cytoplasm. Phosphorylation by the IKK complex",association
R6749 T8614 T8615 themeOf association,necessary
R675 T930 T932 themeOf activation,impaired
R6750 T8634 T8636 themeOf CD40,associated
R6751 T8618 T8621 themeOf CD40,recruited
R6752 T8636 T8637 themeOf associated,reduced
R6753 T8638 T8640 themeOf CD40,immunoprecipitation
R6755 T8639 T8641 themeOf anti-HOIP antibody,recognized
R6759 T8640 T8642 themeOf immunoprecipitation,decrease
R676 T933 T936 themeOf IκB kinase proteins,Recruitment
R6761 T8653 T8658 themeOf TRAF2,association
R677 T934 T936 themeOf CD40,Recruitment
R6775 T8654 T8659 themeOf TRAF3,association
R678 T935 T937 themeOf NF-κB,activation
R6781 T8655 T8660 themeOf cIAP1,association
R6786 T8657 T8658 themeOf CD40,association
R679 T938 T939 themeOf HOIP,expression
R6790 T8657 T8659 themeOf CD40,association
R6792 T8657 T8660 themeOf CD40,association
R6793 T8661 T8663 themeOf IKKα/β and IKKγ,detectable
R6794 T8662 T8664 themeOf CD40,detectable
R6797 T8666 T8668 themeOf IKK,recruitment
R6802 T8667 T8669 themeOf HOIP,expression
R6803 T8669 T8670 themeOf expression,absence
R6804 T8671 T8673 themeOf NF-κB,recruitment
R6809 T8672 T8673 themeOf CD40,recruitment
R6812 T8679 T8682 themeOf IKKγ,recruitment
R6815 T8680 T8682 themeOf CD40,recruitment
R6817 T8681 T8683 themeOf HOIP,requires
R6819 T8682 T8683 causeOf recruitment,requires
R6820 T8687 T8688 themeOf IKKγ,reduced
R6824 T8689 T8691 themeOf CD40,associated
R6828 T8690 T8691 themeOf IKKγ,associated
R6831 T8690 T8692 themeOf IKKγ,phosphorylation
R6832 T8693 T8695 themeOf IKKγ,association
R6838 T8694 T8695 themeOf CD40,association
R9092 T11649 T11653 causeOf CD40,upregulation
R9099 T11650 T11652 themeOf CD80,expression
R9101 T11652 T11653 themeOf expression,upregulation
R9104 T11656 T11658 themeOf IKK complex,recruitment
R9105 T11657 T11658 themeOf CD40,recruitment
R9106 T11658 T11659 themeOf recruitment,promoting
R9108 T11660 T11664 causeOf CD40,induced
R9109 T11661 T11662 themeOf NF-κB,activation
R9110 T11662 T11663 themeOf activation,dependent
R9113 T11662 T11664 themeOf activation,induced
R9114 T11664 T11665 themeOf induced,activation
R9115 T11667 T11669 themeOf JNK,activation
R9116 T11673 T11675 themeOf IKKγ,interact
R9117 T11674 T11676 themeOf NF-κB,activation
R9118 T11708 T11710 themeOf HOIP,recruitment
R9119 T11677 T11679 themeOf IKKγ,recruitment
R9120 T11713 T11717 themeOf ubiquitin ligase,generates
R9121 T11678 T11679 themeOf CD40,recruitment
R9122 T11715 T11717 themeOf polyubiquitin chains,generates
R9123 T11715 T11718 themeOf polyubiquitin chains,bind
R9124 T11715 T11720 themeOf polyubiquitin chains,associate
R9125 T11716 T11719 themeOf CD40,associate
R9126 T11716 T11720 themeOf CD40,associate
R9127 T11679 T11680 themeOf recruitment,mediates
R9128 T11721 T11726 themeOf TRAF2,link
R9129 T11722 T11726 themeOf HOIP,link
R9130 T11682 T11685 themeOf IKKγ,immunoprecipitates
R9131 T11724 T11727 themeOf TRAF6,dependent
R9132 T11725 T11728 themeOf TRAF2,dependent
R9133 T11683 T11685 themeOf CD40,immunoprecipitates
R9134 T11729 T11737 themeOf TRAF,deficient
R9135 T11730 T11738 themeOf NF-κB,activation
R9136 T11684 T11686 themeOf IKKγ,enhance
R9137 T11731 T11738 causeOf CD40,activation
R9138 T11732 T11739 causeOf TRAF2,mediated
R9139 T11687 T11690 themeOf IKK complex,recruitment
R9140 T11733 T11740 causeOf TRAF6,mediated
R9141 T11734 T11741 themeOf JNK,activation
R9142 T11735 T11741 causeOf CD40,activation
R9143 T11736 T11742 causeOf TRAF6,dependent
R9144 T11738 T11739 themeOf activation,mediated
R9145 T11738 T11740 themeOf activation,mediated
R9146 T11688 T11690 themeOf CD40,recruitment
R9147 T11741 T11742 themeOf activation,dependent
R9148 T11689 T11691 themeOf CD40,recruited
R9149 T11743 T11750 causeOf CD40,mediated
R9150 T11743 T11751 causeOf CD40,mediated
R9151 T11744 T11748 themeOf NF-κB,activation
R9152 T11690 T11692 themeOf recruitment,mediates
R9153 T11745 T11749 themeOf JNK,activation
R9154 T11695 T11697 themeOf IKK complex,recruitment
R9155 T11748 T11750 themeOf activation,mediated
R9156 T11749 T11751 themeOf activation,mediated
R9157 T11696 T11697 themeOf CD40,recruitment
R9158 T11752 T11759 causeOf CD40,activation
R9159 T11752 T11760 causeOf CD40,activation
R9160 T11753 T11759 themeOf NF-κB,activation
R9161 T11699 T11703 themeOf IKKγ,binding
R9162 T11754 T11760 themeOf JNK,activation
R9163 T11701 T11704 themeOf IKKγ,recruit
R9164 T11761 T11763 themeOf TRAF6,bind
R9165 T11762 T11763 themeOf CD40,bind
R9166 T11702 T11705 themeOf CD40,complex
R9167 T11765 T11769 themeOf TRAF2,associated
R9168 T11706 T11707 themeOf CD40,recruitment
R9169 T11768 T11769 themeOf Toll-like receptors,associated
R9170 T11770 T11775 themeOf HOIP,interacts
R9171 T11770 T11776 themeOf HOIP,interacts
R9173 T11772 T11775 themeOf SHARPIN,interacts
R9174 T11774 T11776 themeOf RH,interacts
R9175 T11778 T11779 themeOf immunoglobulin,production
R9177 T11782 T11783 themeOf HOIP,regulation
R9362 T12050 T12051 themeOf CD154,expressing