IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell (0092-8674) 2007 imex curation Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer reedoffice@burnham.org 23-MAY-2007: Contacted by JYOTI. Returned mail. Only protein-protein interactions 24 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis reedoffice@burnham.org Accepted 2007-MAY-30 by DAVET. 23-MAY-2007: Contacted by JYOTI. Returned mail. Cell (0092-8674) 2007 Only protein-protein interactions 24 imex curation Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell (0092-8674) 2007 imex curation Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer reedoffice@burnham.org 23-MAY-2007: Contacted by JYOTI. Returned mail. Only protein-protein interactions 24 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis The range of the Bcl-2 Delta loop deletion was found in Fang et al. 1998 Cancer Research 58 3202-3208 reedoffice@burnham.org Accepted 2007-MAY-30 by DAVET. 23-MAY-2007: Contacted by JYOTI. Returned mail. Cell (0092-8674) 2007 Only protein-protein interactions 24 imex curation Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell (0092-8674) 2007 imex curation Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer reedoffice@burnham.org 23-MAY-2007: Contacted by JYOTI. Returned mail. Only protein-protein interactions 24 human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 confocal microscopy confocal microscopy antibody detection identification by antibody antibody detection Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis reedoffice@burnham.org Accepted 2007-MAY-30 by DAVET. 23-MAY-2007: Contacted by JYOTI. Returned mail. Cell (0092-8674) 2007 Only protein-protein interactions 24 imex curation Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell (0092-8674) 2007 imex curation Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer reedoffice@burnham.org 23-MAY-2007: Contacted by JYOTI. Returned mail. Only protein-protein interactions 24 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis reedoffice@burnham.org Accepted 2007-MAY-30 by DAVET. 23-MAY-2007: Contacted by JYOTI. Returned mail. Cell (0092-8674) 2007 Only protein-protein interactions 24 imex curation Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell (0092-8674) 2007 imex curation Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer reedoffice@burnham.org 23-MAY-2007: Contacted by JYOTI. Returned mail. Only protein-protein interactions 24 in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis reedoffice@burnham.org Accepted 2007-MAY-30 by DAVET. 23-MAY-2007: Contacted by JYOTI. Returned mail. Cell (0092-8674) 2007 Only protein-protein interactions 24 imex curation Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell (0092-8674) 2007 imex curation Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer reedoffice@burnham.org 23-MAY-2007: Contacted by JYOTI. Returned mail. Only protein-protein interactions 24 human-thp-1 Homo sapiens acute monocytic leukemia cells thp-1 Human acute monocytic leukemia cell line anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Bruey JM., Bruey-Sedano N., Luciano F., Zhai D., Balpai R., Xu C., Kress CL., Bailly-Maitre B., Li X., Osterman A., Matsuzawa S., Terskikh AV., Faustin B., Reed JC. Apoptosis - Interactions involving proteins with a function related to apoptosis reedoffice@burnham.org Accepted 2007-MAY-30 by DAVET. 23-MAY-2007: Contacted by JYOTI. Returned mail. Cell (0092-8674) 2007 Only protein-protein interactions 24 imex curation b2cl1_human Bcl-2-like protein 1 BCL2L1 BCL2L BCLX Apoptosis regulator Bcl-X protein protein human Homo sapiens Human MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK E09D3CDD851AE9BE nlrc4_human NLR family CARD domain-containing protein 4 NLRC4 CLAN CLAN1 UNQ6189/PRO20215 CARD12 IPAF Caspase recruitment domain-containing protein 12 Ice protease-activating factor CARD, LRR, and NACHT-containing protein protein protein human Homo sapiens Human MNFIKDNSRALIQRMGMTVIKQITDDLFVWNVLNREEVNIICCEKVEQDAARGIIHMILKKGSESCNLFLKSLKEWNYPLFQDLNGQSLFHQTSEGDLDDLAQDLKDLYHTPSFLNFYPLGEDIDIIFNLKSTFTEPVLWRKDQHHHRVEQLTLNGLLQALQSPCIIEGESGKGKSTLLQRIAMLWGSGKCKALTKFKFVFFLRLSRAQGGLFETLCDQLLDIPGTIRKQTFMAMLLKLRQRVLFLLDGYNEFKPQNCPEIEALIKENHRFKNMVIVTTTTECLRHIRQFGALTAEVGDMTEDSAQALIREVLIKELAEGLLLQIQKSRCLRNLMKTPLFVVITCAIQMGESEFHSHTQTTLFHTFYDLLIQKNKHKHKGVAASDFIRSLDHCGDLALEGVFSHKFDFELQDVSSVNEDVLLTTGLLCKYTAQRFKPKYKFFHKSFQEYTAGRRLSSLLTSHEPEEVTKGNGYLQKMVSISDITSTYSSLLRYTCGSSVEATRAVMKHLAAVYQHGCLLGLSIAKRPLWRQESLQSVKNTTEQEILKAININSFVECGIHLYQESTSKSALSQEFEAFFQGKSLYINSGNIPDYLFDFFEHLPNCASALDFIKLDFYGGAMASWEKAAEDTGGIHMEEAPETYIPSRAVSLFFNWKQEFRTLEVTLRDFSKLNKQDIRYLGKIFSSATSLRLQIKRCAGVAGSLSLVLSTCKNIYSLMVEASPLTIEDERHITSVTNLKTLSIHDLQNQRLPGGLTDSLGNLKNLTKLIMDNIKMNEEDAIKLAEGLKNLKKMCLFHLTHLSDIGEGMDYIVKSLSSEPCDLEEIQLVSCCLSANAVKILAQNLHNLVKLSILDLSENYLEKDGNEALHELIDRMNVLEQLTALMLPWGCDVQGSLSSLLKHLEEVPQLVKLGLKNWRLTDTEIRILGAFFGKNPLKNFQQLNLAGNRVSSDGWLAFMGVFENLKQLVFFDFSTKEFLPDPALVRKLSQVLSKLTFLQEARLVGWQFDDDDLSVITGAFKLVTA 49378DBB54938E0F casp5_human Caspase-5 CASP5 ICH3 Protease ICH-3 Protease TY ICE(rel)-III protein protein human Homo sapiens Human MAEDSGKKKRRKNFEAMFKGILQSGLDNFVINHMLKNNVAGQTSIQTLVPNTDQKSTSVKKDNHKKKTVKMLEYLGKDVLHGVFNYLAKHDVLTLKEEEKKKYYDTKIEDKALILVDSLRKNRVAHQMFTQTLLNMDQKITSVKPLLQIEAGPPESAESTNILKLCPREEFLRLCKKNHDEIYPIKKREDRRRLALIICNTKFDHLPARNGAHYDIVGMKRLLQGLGYTVVDEKNLTARDMESVLRAFAARPEHKSSDSTFLVLMSHGILEGICGTAHKKKKPDVLLYDTIFQIFNNRNCLSLKDKPKVIIVQACRGEKHGELWVRDSPASLALISSQSSENLEADSVCKIHEEKDFIAFCSSTPHNVSWRDRTRGSIFITELITCFQKYSCCCHLMEIFRKVQKSFEVPQAKAQMPTIERATLTRDFYLFPGN DIP protein P51878 original sequence version: 126 C5257C2BF15EB6D5 nlrp1_human NACHT, LRR and PYD domains-containing protein 1 NLRP1 CARD7 DEFCAP KIAA0926 NAC NALP1 Death effector filament-forming ced-4-like apoptosis protein Nucleotide-binding domain and caspase recruitment domain Caspase recruitment domain-containing protein 7 protein protein human Homo sapiens Human MAGGAWGRLACYLEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSGMEVASYLVAQYGEQRAWDLALHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAVLMPWIHELPAGCTQGSERRVLRQLPDTSGRRWREISASLLYQALPSSPDHESPSQESPNAPTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDETSGIYYTEIREREREKSEKGRPPWAAVVGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQLLLLQRPHPRSQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEAWGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDGVDEPGWVLQEPSSELCLHWSQPQPADALLGSLLGKTILPEASFLITARTTALQNLIPSLEQARWVEVLGFSESSRKEYFYRYFTDERQAIRAFRLVKSNKELWALCLVPWVSWLACTCLMQQMKRKEKLTLTSKTTTTLCLHYLAQALQAQPLGPQLRDLCSLAAEGIWQKKTLFSPDDLRKHGLDGAIISTFLKMGILQEHPIPLSYSFIHLCFQEFFAAMSYVLEDEKGRGKHSNCIIDLEKTLEAYGIHGLFGASTTRFLLGLLSDEGEREMENIFHCRLSQGRNLMQWVPSLQLLLQPHSLESLHCLYETRNKTFLTQVMAHFEEMGMCVETDMELLVCTFCIKFSRHVKKLQLIEGRQHRSTWSPTMVVLFRWVPVTDAYWQILFSVLKVTRNLKELDLSGNSLSHSAVKSLCKTLRRPRCLLETLRLAGCGLTAEDCKDLAFGLRANQTLTELDLSFNVLTDAGAKHLCQRLRQPSCKLQRLQLVSCGLTSDCCQDLASVLSASPSLKELDLQQNNLDDVGVRLLCEGLRHPACKLIRLGLDQTTLSDEMRQELRALEQEKPQLLIFSRRKPSVMTPTEGLDTGEMSNSTSSLKRQRLGSERAASHVAQANLKLLDVSKIFPIAEIAEESSPEVVPVELLCVPSPASQGDLHTKPLGTDDDFWGPTGPVATEVVDKEKNLYRVHFPVAGSYRWPNTGLCFVMREAVTVEIEFCVWDQFLGEINPQHSWMVAGPLLDIKAEPGAVEAVHLPHFVALQGGHVDTSLFQMAHFKEEGMLLEKPARVELHHIVLENPSFSPLGVLLKMIHNALRFIPVTSVVLLYHRVHPEEVTFHLYLIPSDCSIRKAIDDLEMKFQFVRIHKPPPLTPLYMGCRYTVSGSGSGMLEILPKELELCYRSPGEDQLFSEFYVGHLGSGIRLQVKDKKDETLVWEALVKPGDLMPATTLIPPARIAVPSPLDAPQLLHFVDQYREQLIARVTSVEVVLDKLHGQVLSQEQYERVLAENTRPSQMRKLFSLSQSWDRKCKDGLYQALKETHPHLIMELWEKGSKKGLLPLSS DIP protein Q9C000 original sequence version: 87 DIP protein Q9C000 original sequence version: 114 DIP protein Q9C000 original sequence version: 131 438F0DCE45C2562D bcl2_human Apoptosis regulator Bcl-2 BCL2 protein protein human Homo sapiens Human MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK DIP protein P10415 original sequence version: 148 DIP protein P10415 original sequence version: 125 DIP protein P10415 original sequence version: 151 DIP protein P10415 original sequence version: 204 DIP protein P10415 original sequence version: 149 3C49F2B714DC9CCB asc_human Apoptosis-associated speck-like protein containing a CARD PYD and CARD domain-containing protein Target of methylation-induced silencing 1 Caspase recruitment domain-containing protein 5 ASC CARD5 TMS1 PYCARD protein protein human Homo sapiens Human MGRARDAILDALENLTAEELKKFKLKLLSVPLREGYGRIPRGALLSMDALDLTDKLVSFYLETYGAELTANVLRDMGLQEMAGQLQAATHQGSGAAPAGIQAPPQSAAKPGLHFIDQHRAALIARVTNVEWLLDALYGKVLTDEQYQAVRAEPTNPSKMRKLFSFTPAWNWTCKDLLLQALRESQSYLVEDLERS DIP protein Q9ULZ3 original sequence version: 116 455987286586F46A casp1_human Caspase-1 Interleukin-1 beta convertase Interleukin-1 beta-converting enzyme p45 CASP1 IL1BC IL1BCE protein protein human Homo sapiens Human MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALIDSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQDNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH DIP protein P29466 original sequence version: 155 DIP protein P29466-1 original sequence version: 169 DIP protein P29466 original sequence version: 169 DIP protein P29466 original sequence version: 163 DIP protein P29466 original sequence version: 101 DIP protein P29466 original sequence version: 160 DIP protein P29466 original sequence version: 184 ABF33CF33CC71584 bcl2-nalp1 4 11 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 A NALP1 was found to associate with Bcl-2 and Bcl-XL by coimmunoprecipitation (coIP) experiments using lysates prepared from transfected HEK293T cells expressing epitopetagged proteins. Q9C000; P10415 transfection bcl2l1-nalp1 4 10 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 A NALP1 was found to associate with Bcl-2 and Bcl-XL by coimmunoprecipitation (coIP) experiments using lysates prepared from transfected HEK293T cells expressing epitopetagged proteins. Q9C000; Q07817 transfection nalp1-bcl2l1 4 7 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 B NALP1 was found to associate with Bcl-2 and Bcl-XL by coimmunoprecipitation (coIP) experiments using lysates prepared from transfected HEK293T cells expressing epitopetagged proteins. Q9C000; Q07817 transfection bcl2-nalp1-1 5 10 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 C Of the six human antiapoptotic Bcl-2-family proteins, only Bcl-2 and Bcl-XL associated with NALP1. In contrast, Mcl-1, Bcl-W, Bfl-1, and Bcl-B did not associate with NALP1 (Figures 1A and 1B), nor did various proapoptotic Bcl-2-family proteins, including Bax, Bak, Bid, and Bcl-G (Figure 1B). Similar conclusions were reached using in vitro protein-binding assays where NALP1-containing cell lysates were incubated with bacteria-produced GST-fusion proteins bcl2l2l1-nalp1 5 10 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 C Of the six human antiapoptotic Bcl-2-family proteins, only Bcl-2 and Bcl-XL associated with NALP1. In contrast, Mcl-1, Bcl-W, Bfl-1, and Bcl-B did not associate with NALP1 (Figures 1A and 1B), nor did various proapoptotic Bcl-2-family proteins, including Bax, Bak, Bid, and Bcl-G (Figure 1B). Similar conclusions were reached using in vitro protein-binding assays where NALP1-containing cell lysates were incubated with bacteria-produced GST-fusion proteins bcl2l1l2-nalp1 1 7 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 D However, among these proteins tested, only NALP1 associated with Bcl-XL and Bcl-2 Q9C000; Q07817 transfection bcl2-nalp1-2 1 10 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 11 unspecified role unspecified role bait bait physical association physical association 1 E However, among these proteins tested, only NALP1 associated with Bcl-XL and Bcl-2 Q9C000; Q07817 transfection nalp1-pycard 6 10 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey 11 inhibitor inhibitor prey prey association association 2 B, 5 F When endogenous NALP1 was immunoprecipitated from untreated macrophages using anti-NALP1 antibody, endogenous Bcl-2 and Bcl-XL were associated with NALP1-containing immune complexes, while ASC was not (Figure 2B). These macrophages evidently contain more Bcl-2 than Bcl-XL, which possibly accounts for the clearer association of Bcl-2 with NALP1 immunoprecipitates when compared to Bcl-XL. In contrast, when immunoprecipitated from MDP/ATP-treated (Figure 2B) or LPS/ATP-treated (Figure S1) macrophages, ASC was associated with NALP1-containing immune complexes, while Bcl-2 and Bcl-XL were not. This interaction occurred when cells were cultured with MDP/ATP, MDP-LD or LPS. P10415 infection nalp1-bcl2 6 11 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait association association 2 B When endogenous NALP1 was immunoprecipitated from untreated macrophages using anti-NALP1 antibody, endogenous Bcl-2 and Bcl-XL were associated with NALP1-containing immune complexes, while ASC was not (Figure 2B). This interaction did not occur when cells were cultured with MDP/ATP. bcl2-nalp1-4 6 10 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait physical association physical association 2 C This interaction does not occur in LPS/ATP treated cells. pycard-nalp1 6 12 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association 2 D This interaction occurs in cells treated with LPS/ATP. when immunoprecipitated from MDP/ATP-treated (Figure 2B) or LPS/ATP-treated (Figure S1) macrophages, ASC was associated with NALP1-containing immune complexes nalp1-bcl2-2 6 12 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey association association S1 These interactions occur in cells treated with LPS/ATP. nalp1-bcl2-1 6 11 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association S1 casp1-nalp1 2 10 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association S2 P29466; Q9C000 transfection casp5-nalp1 2 9 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association S2 Q9C000; P51878 transfection casp1-card12 2 13 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association S2 Q9NPP4; P29466 transfection nalp1-pycard-1 2 12 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 inhibitor inhibitor unspecified role unspecified role region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 3 C When active MDP-LD (but not MDP-DD) was added to extracts derived from HEK293T cells transfected with plasmids encoding GFP-tagged ASC and epitope-tagged NALP1, we observed that GFP-ASC inducibly associated with NALP1 (Figures 3C and 3D). Addition of Bcl-XL or Bcl-2 to the extracts prevented GFP-ASC from binding to NALP1. Q9ULZ3; Q9C000; Q07817 transfection The interaction did not occur in the presence of MDP-DD. The presence of GST-Bl-xL inhibited the interaction. nalp1-pycard-2 2 11 inhibitor inhibitor unspecified role unspecified role region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 12 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 3 D When active MDP-LD (but not MDP-DD) was added to extracts derived from HEK293T cells transfected with plasmids encoding GFP-tagged ASC and epitope-tagged NALP1, we observed that GFP-ASC inducibly associated with NALP1 (Figures 3C and 3D). Addition of Bcl-XL or Bcl-2 to the extracts prevented GFP-ASC from binding to NALP1. Q9ULZ3; Q9C000; P10415 transfection The interaction did not occur in the presence of MDP-DD. The presence of Bcl-2-GST inhibited the interaction. The presence of Bcl-B-GST did not inhibit the interaction. bcl2-nalp1-5 2 10 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 11 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 4 A Q9C000; P10415 transfection Removal of the loop from Bcl-2 or Bcl-XL abolished interaction with NALP1 (Figures 4A and S7A). In contrast, deleting BH3 or BH4 domains from Bcl-XL did not impair binding to NALP1, as determined by coIP experiments bcl2l2l1-nalp1-1 2 7 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 4 A Q9C000; Q07817 transfection Removal of the loop from Bcl-2 or Bcl-XL abolished interaction with NALP1 (Figures 4A and S7A). In contrast, deleting BH3 or BH4 domains from Bcl-XL did not impair binding to NALP1, as determined by coIP experiments bcl2l2l1-nalp1-2 2 10 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain physical association physical association S7B Q9C000; Q07817 transfection Removal of the loop from Bcl-2 or Bcl-XL abolished interaction with NALP1 (Figures 4A and S7A). In contrast, deleting BH3 or BH4 domains from Bcl-XL did not impair binding to NALP1, as determined by coIP experiments bcl2l2l1-nalp1-3 2 7 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 4 D Q9C000; Q07817 transfection we attempted to map the region of NALP1 required for binding Bcl-XL. These experiments demonstrated that the LRRs (but not the CARD or FIIND domains)of NALP1 are necessary, but insufficient, for binding Bcl-XL bcl2-nalp1-6 3 11 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role neutral component neutral component region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization S8 These experiments demonstrated that the LRRs (but not the CARD or FIIND domains)of NALP1 are necessary, but insufficient, for binding Bcl-XL (Figures 4D, 4E, S7C, and S7D). These protein-interaction studies were performed by coIP using cell lysates and were independently confirmed by immunofluorescence confocal microscopy analysis of intact cells, where fulllength NALP1 but not NALP1DLRR was shown to redistribute from a diffuse cytosolic to an organellar location when coexpressed with Bcl-2 bcl2-nalp1-7 3 11 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain colocalization colocalization S8 These experiments demonstrated that the LRRs (but not the CARD or FIIND domains)of NALP1 are necessary, but insufficient, for binding Bcl-XL (Figures 4D, 4E, S7C, and S7D). These protein-interaction studies were performed by coIP using cell lysates and were independently confirmed by immunofluorescence confocal microscopy analysis of intact cells, where fulllength NALP1 but not NALP1DLRR was shown to redistribute from a diffuse cytosolic to an organellar location when coexpressed with Bcl-2