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/ http://www.ebi.ac.uk/ sun-2012-6 Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Sun L., Wang H., Wang Z., He S., Chen S., Liao D., Wang L., Yan J., Liu W., Lei X., Wang X. Cells treated with necrosis inducers in the presence of necrosulfonamide. leixiaoguang@nibs.ac.cn, wangxiaodong@nibs.ac.cn Cell (0092-8674) 2012 Only protein-protein interactions imex curation sun-2012-3 Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Sun L., Wang H., Wang Z., He S., Chen S., Liao D., Wang L., Yan J., Liu W., Lei X., Wang X. Cells treated with necrosis inducers in the presence of necrosulfonamide. leixiaoguang@nibs.ac.cn, wangxiaodong@nibs.ac.cn Cell (0092-8674) 2012 Only protein-protein interactions imex curation sun-2012-1 Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. human-ht_29 Homo sapiens HT-29 caucasian women with colon adenocarcinoma ht_29 HT-29 confirmed as human with IEF of AST, LDH established from the primary tumor of a 44-year-old Caucasian woman with colon adenocarcinoma in 1964; described to be heterotransplantable forming well-differentiated grade I tumors anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Sun L., Wang H., Wang Z., He S., Chen S., Liao D., Wang L., Yan J., Liu W., Lei X., Wang X. Cells treated with necrosis inducers in the presence of necrosulfonamide. leixiaoguang@nibs.ac.cn, wangxiaodong@nibs.ac.cn Cell (0092-8674) 2012 Only protein-protein interactions imex curation sun-2012-5 Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Sun L., Wang H., Wang Z., He S., Chen S., Liao D., Wang L., Yan J., Liu W., Lei X., Wang X. Cells treated with necrosis inducers in the presence of necrosulfonamide. leixiaoguang@nibs.ac.cn, wangxiaodong@nibs.ac.cn Cell (0092-8674) 2012 Only protein-protein interactions imex curation sun-2012-2 Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti tag coip anti tag coimmunoprecipitation anti tag coip sequence tag sequence tag identification sequence tag Sun L., Wang H., Wang Z., He S., Chen S., Liao D., Wang L., Yan J., Liu W., Lei X., Wang X. Cells treated with necrosis inducers in the presence of necrosulfonamide. leixiaoguang@nibs.ac.cn, wangxiaodong@nibs.ac.cn Cell (0092-8674) 2012 Only protein-protein interactions imex curation sun-2012-4 Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. in vitro In vitro comigration in sds comigration in sds page comigration in sds western blot western blot Immuno blot Immuno blot Sun L., Wang H., Wang Z., He S., Chen S., Liao D., Wang L., Yan J., Liu W., Lei X., Wang X. Cells treated with necrosis inducers in the presence of necrosulfonamide. leixiaoguang@nibs.ac.cn, wangxiaodong@nibs.ac.cn Cell (0092-8674) 2012 Only protein-protein interactions imex curation sun-2012-7 Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Sun L., Wang H., Wang Z., He S., Chen S., Liao D., Wang L., Yan J., Liu W., Lei X., Wang X. Cells treated with necrosis inducers in the presence of necrosulfonamide. leixiaoguang@nibs.ac.cn, wangxiaodong@nibs.ac.cn Cell (0092-8674) 2012 Only protein-protein interactions imex curation ripk3_human Receptor-interacting serine/threonine-protein kinase 3 RIPK3 RIP3 RIP-like protein kinase 3 Receptor-interacting protein 3 protein protein human Homo sapiens Human MSCVKLWPSGAPAPLVSIEELENQELVGKGGFGTVFRAQHRKWGYDVAVKIVNSKAISREVKAMASLDNEFVLRLEGVIEKVNWDQDPKPALVTKFMENGSLSGLLQSQCPRPWPLLCRLLKEVVLGMFYLHDQNPVLLHRDLKPSNVLLDPELHVKLADFGLSTFQGGSQSGTGSGEPGGTLGYLAPELFVNVNRKASTASDVYSFGILMWAVLAGREVELPTEPSLVYEAVCNRQNRPSLAELPQAGPETPGLEGLKELMQLCWSSEPKDRPSFQECLPKTDEVFQMVENNMNAAVSTVKDFLSQLRSSNRRFSIPESGQGGTEMDGFRRTIENQHSRNDVMVSEWLNKLNLEEPPSSVPKKCPSLTKRSRAQEEQVPQAWTAGTSSDSMAQPPQTPETSTFRNQMPSPTSTGTPSPGPRGNQGAERQGMNWSCRTPEPNPVTGRPLVNIYNCSGVQVGDNNYLTMQQTTALPTWGLAPSGKGRGLQHPPPVGSQEGPKDPEAWSRPQGWYNHSGK mlkl_human Mixed lineage kinase domain-like protein MLKL protein protein human Homo sapiens Human MENLKHIITLGQVIHKRCEEMKYCKKQCRRLGHRVLGLIKPLEMLQDQGKRSVPSEKLTTAMNRFKAALEEANGEIEKFSNRSNICRFLTASQDKILFKDVNRKLSDVWKELSLLLQVEQRMPVSPISQGASWAQEDQQDADEDRRAFQMLRRDNEKIEASLRRLEINMKEIKETLRQYLPPKCMQEIPQEQIKEIKKEQLSGSPWILLRENEVSTLYKGEYHRAPVAIKVFKKLQAGSIAIVRQTFNKEIKTMKKFESPNILRIFGICIDETVTPPQFSIVMEYCELGTLRELLDREKDLTLGKRMVLVLGAARGLYRLHHSEAPELHGKIRSSNFLVTQGYQVKLAGFELRKTQTSMSLGTTREKTDRVKSTAYLSPQELEDVFYQYDVKSEIYSFGIVLWEIATGDIPFQGCNSEKIRKLVAVKRQQEPLGEDCPSELREIIDECRAHDPSVRPSVDEILKKLSTFSK mlkl_mouse Mixed lineage kinase domain-like protein Mlkl protein protein mouse Mus musculus Mouse MDKLGQIIKLGQLIYEQCEKMKYCRKQCQRLGNRVHGLLQPLQRLQAQGKKNLPDDITAALGRFDEVLKEANQQIEKFSKKSHIWKFVSVGNDKILFHEVNEKLRDVWEELLLLLQVYHWNTVSDVSQPASWQQEDRQDAEEDGNENMKVILMQLQISVEEINKTLKQCSLKPTQEIPQDLQIKEIPKEHLGPPWTKLKTSKMSTIYRGEYHRSPVTIKVFNNPQAESVGIVRFTFNDEIKTMKKFDSPNILRIFGICIDQTVKPPEFSIVMEYCELGTLRELLDREKDLTMSVRSLLVLRAARGLYRLHHSETLHRNISSSSFLVAGGYQVKLAGFELSKTQNSISRTAKSTKAERSSSTIYVSPERLKNPFCLYDIKAEIYSFGIVLWEIATGKIPFEGCDSKKIRELVAEDKKQEPVGQDCPELLREIINECRAHEPSQRPSVDGRSLSGRERILERLSAVEESTDKKV ripk3_mouse Receptor-interacting serine/threonine-protein kinase 3 Ripk3 Rip3 RIP-like protein kinase 3 Receptor-interacting protein 3 protein protein mouse Mus musculus Mouse MSSVKLWPTGASAVPLVSREELKKLEFVGKGGFGVVFRAHHRTWNHDVAVKIVNSKKISWEVKAMVNLRNENVLLLLGVTEDLQWDFVSGQALVTRFMENGSLAGLLQPECPRPWPLLCRLLQEVVLGMCYLHSLNPPLLHRDLKPSNILLDPELHAKLADFGLSTFQGGSQSGSGSGSGSRDSGGTLAYLDPELLFDVNLKASKASDVYSFGILVWAVLAGREAELVDKTSLIRETVCDRQSRPPLTELPPGSPETPGLEKLKELMIHCWGSQSENRPSFQDCEPKTNEVYNLVKDKVDAAVSEVKHYLSQHRSSGRNLSAREPSQRGTEMDCPRETMVSKMLDRLHLEEPSGPVPGKCPERQAQDTSVGPATPARTSSDPVAGTPQIPHTLPFRGTTPGPVFTETPGPHPQRNQGDGRHGTPWYPWTPPNPMTGPPALVFNNCSEVQIGNYNSLVAPPRTTASSSAKYDQAQFGRGRGWQPFHK necrosulfonamide small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) ripk1_human Receptor-interacting serine/threonine-protein kinase 1 RIPK1 RIP Cell death protein RIP RIP1 Receptor-interacting protein 1 protein protein human Homo sapiens Human MQPDMSLNVIKMKSSDFLESAELDSGGFGKVSLCFHRTQGLMIMKTVYKGPNCIEHNEALLEEAKMMNRLRHSRVVKLLGVIIEEGKYSLVMEYMEKGNLMHVLKAEMSTPLSVKGRIILEIIEGMCYLHGKGVIHKDLKPENILVDNDFHIKIADLGLASFKMWSKLNNEEHNELREVDGTAKKNGGTLYYMAPEHLNDVNAKPTEKSDVYSFAVVLWAIFANKEPYENAICEQQLIMCIKSGNRPDVDDITEYCPREIISLMKLCWEANPEARPTFPGIEEKFRPFYLSQLEESVEEDVKSLKKEYSNENAVVKRMQSLQLDCVAVPSSRSNSATEQPGSLHSSQGLGMGPVEESWFAPSLEHPQEENEPSLQSKLQDEANYHLYGSRMDRQTKQQPRQNVAYNREEERRRRVSHDPFAQQRPYENFQNTEGKGTAYSSAASHGNAVHQPSGLTSQPQVLYQNNGLYSSHGFGTRPLDPGTAGPRVWYRPIPSHMPSLHNIPVPETNYLGNTPTMPFSSLPPTDESIKYTIYNSTGIQIGAYNYMEIGGTSSSLLDSTNTNFKEEPAAKYQAIFDNTTSLTDKHLDPIRENLGKHWKNCARKLGFTQSQIDEIDHDYERDGLKEKVYQMLQKWVMREGIKGATVGKLAQALHQCSRIDLLSSLIYVSQN ripk3-mlkl-10 1266855 n/a 1266862 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 false n/a 1266863 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 false physical association physical association false false false Fig. S5D mlkl-ripk3 1266855 n/a 1266863 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 false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1266862 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q9Y572:p.Ser227Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q9Y572:p.Asp142Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false 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 false physical association physical association false false false Fig. 5B, 7C. ripk3-mlkl-4 1266855 n/a 1266862 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 false n/a 1266863 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 false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 1266888 1266884 physical association physical association false false false Fig. 5C. mlkl-ripk3-2 1266855 n/a 1266864 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 false n/a 1266865 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 false physical association physical association false false false S6 Mouse RIP3 only binds to mouse MLKL, but not the human homolog. necrosulfonam-mlkl-1 1266856 n/a 1266863 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 n/a 1266866 unspecified role unspecified role bait bait region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1266862 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false 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 false human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 association association false false false Fig. 3C Non-biotin-labeled necrosulfonamide competed off MLKL and RIP3 binding to the beads. endogenous RIP3 was also pulled down with MLKL before necrosis induction, confirming there was a basal level of MLKL/RIP3 interaction in living cells. As necrosis progressed, less MLKL/RIP3 bound to the biotin-NSA/streptavidin-conjugated beads. necrosulfonamid-mlkl 1266856 n/a 1266863 unspecified role unspecified role prey prey n_terminus myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false Q8NB16:p.Cys86Ser mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 n/a 1266866 unspecified role unspecified role bait bait region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false Figs. 3D, 4B, 4D, Supp. Figs. 4C, 5E Mouse MLKL does not bind necrosulfonamide. necrosulfonam-mlkl-2 1266856 n/a 1266866 unspecified role unspecified role bait bait region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1266863 unspecified role unspecified role prey prey human-ht_29 Homo sapiens HT-29 caucasian women with colon adenocarcinoma ht_29 HT-29 confirmed as human with IEF of AST, LDH established from the primary tumor of a 44-year-old Caucasian woman with colon adenocarcinoma in 1964; described to be heterotransplantable forming well-differentiated grade I tumors n/a 1266862 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 false region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false human-ht_29 Homo sapiens HT-29 caucasian women with colon adenocarcinoma ht_29 HT-29 confirmed as human with IEF of AST, LDH established from the primary tumor of a 44-year-old Caucasian woman with colon adenocarcinoma in 1964; described to be heterotransplantable forming well-differentiated grade I tumors association association false false false Supp. Fig. 3B Non-biotin-labeled necrosulfonamide competed off MLKL and RIP3 binding to the beads. endogenous RIP3 was also pulled down with MLKL before necrosis induction, confirming there was a basal level of MLKL/RIP3 interaction in living cells. As necrosis progressed, less MLKL/RIP3 bound to the biotin-NSA/streptavidin-conjugated beads. ripk3-ripk1-2 1266857 n/a 1266862 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 false n/a 1266867 unspecified role unspecified role prey prey physical association physical association false false false Fig. 1G The interaction between RIP1 and RIP3 was enhanced by Necrosulfonamide. ripk3-mlkl-1 1266857 n/a 1266862 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 false n/a 1266863 unspecified role unspecified role prey prey physical association physical association false false false Fig. 2B ripk3-mlkl-2 1266858 n/a 1266863 unspecified role unspecified role prey prey n/a 1266862 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 false n/a 1266867 unspecified role unspecified role prey prey physical association physical association false false false Fig. 2D Necrostatin-1 Necrosulfonamide MLKL knockdown enhanced RIP1/RIP3 interaction, but this interaction was not further enhanced by necrosulfonamide treatment after MLKL knockdown. ripk3-mlkl-9 1266858 n/a 1266862 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 false Q9Y572:p.Lys50Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false n/a 1266863 unspecified role unspecified role prey prey physical association physical association false false false Fig. 5D ripk3-mlkl 1266859 n/a 1266863 unspecified role unspecified role prey prey n/a 1266862 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 false region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Fig. 2A mlkl-necrosulfonamid 1266860 n/a 1266863 unspecified role unspecified role neutral component neutral component n/a 1266866 unspecified role unspecified role neutral component neutral component region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction false false false Fig. 3E Non-biotin-labeled necrosulfonamide competed off MLKL and RIP3 binding to the beads. mlkl-ripk3-1 1266861 n/a 1266863 enzyme target enzyme target substrate substrate neutral component neutral component Q8NB16:p.Thr357_Ser358delinsAlaAla mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false Q8NB16:p.Thr357_Ser358delinsGluAsp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1266862 enzyme enzyme neutral component neutral component region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false 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 false human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 physical association physical association false false false Fig. 5E, 6A, 6C, Supp. Fig. 5A Tandem mass spectrometry of MLKL unambiguously identified threonine 357 and serine 358 as being phosphorylated