MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex. Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex. EMBO J. (0261-4189) 2013 imex curation Sedgwick GG., Hayward DG., Di Fiore B., Pardo M., Yu L., Pines J., Nilsson J. Only protein-protein interactions human-u2-os Human U2-OS osteosarcoma cell line u2-os U2-OS osteocaroma cell line Not in CAPRI and no primary literature reference anti bait coip anti bait coimmunoprecipitation anti bait coip experimental particp experimental participant identification experimental particp Sedgwick GG., Hayward DG., Di Fiore B., Pardo M., Yu L., Pines J., Nilsson J. EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex. Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex. EMBO J. (0261-4189) 2013 imex curation Sedgwick GG., Hayward DG., Di Fiore B., Pardo M., Yu L., Pines J., Nilsson J. Only protein-protein interactions 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 experimental particp experimental participant identification experimental particp Sedgwick GG., Hayward DG., Di Fiore B., Pardo M., Yu L., Pines J., Nilsson J. EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex. Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex. EMBO J. (0261-4189) 2013 imex curation Sedgwick GG., Hayward DG., Di Fiore B., Pardo M., Yu L., Pines J., Nilsson J. Only protein-protein interactions 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 bait coip anti bait coimmunoprecipitation anti bait coip experimental particp experimental participant identification experimental particp Sedgwick GG., Hayward DG., Di Fiore B., Pardo M., Yu L., Pines J., Nilsson J. EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation cdc20_human Cell division cycle protein 20 homolog CDC20 p55CDC protein protein human Homo sapiens Human MAQFAFESDLHSLLQLDAPIPNAPPARWQRKAKEAAGPAPSPMRAANRSHSAGRTPGRTPGKSSSKVQTTPSKPGGDRYIPHRSAAQMEVASFLLSKENQPENSQTPTKKEHQKAWALNLNGFDVEEAKILRLSGKPQNAPEGYQNRLKVLYSQKATPGSSRKTCRYIPSLPDRILDAPEIRNDYYLNLVDWSSGNVLAVALDNSVYLWSASSGDILQLLQMEQPGEYISSVAWIKEGNYLAVGTSSAEVQLWDVQQQKRLRNMTSHSARVGSLSWNSYILSSGSRSGHIHHHDVRVAEHHVATLSGHSQEVCGLRWAPDGRHLASGGNDNLVNVWPSAPGEGGWVPLQTFTQHQGAVKAVAWCPWQSNVLATGGGTSDRHIRIWNVCSGACLSAVDAHSQVCSILWSPHYKELISGHGFAQNQLVIWKYPTMAKVAELKGHTSRVLSLTMSPDGATVASAAADETLRLWRCFELDPARRREREKASAAKSSLIHQGIR DIP protein Q12834 original sequence version: 132 DIP protein Q12834 original sequence version: 74 DIP protein Q12834 original sequence version: 100 DIP protein Q12834 original sequence version: 118 DIP protein Q12834 original sequence version: 125 DIP protein Q12834 original sequence version: 114 DIP protein Q12834 original sequence version: 147 DIP protein Q12834 original sequence version: 162 FD5C967AF84089E8 md2l1_human Mitotic spindle assembly checkpoint protein MAD2A MAD2L1 MAD2 Mitotic arrest deficient 2-like protein 1 protein protein human Homo sapiens Human MALQLSREQGITLRGSAEIVAEFFSFGINSILYQRGIYPSETFTRVQKYGLTLLVTTDLELIKYLNNVVEQLKDWLYKCSVQKLVVVISNIESGEVLERWQFDIECDKTAKDDSAPREKSQKAIQDEIRSVIRQITATVTFLPLLEVSCSFDLLIYTDKDLVVPEKWEESGPQFITNSEEVRLRSFTTTIHKVNSMVAYKIPVND DIP protein Q13257 original sequence version: 133 DIP protein Q13257 original sequence version: 78 DIP protein Q13257 original sequence version: 105 DIP protein Q13257 original sequence version: 120 DIP protein Q13257 original sequence version: 127 DIP protein Q13257 original sequence version: 116 DIP protein Q13257 original sequence version: 150 DIP protein Q13257 original sequence version: 156 DIP protein Q13257 original sequence version: 134 B8DCBF0043836764 scmc1_human Calcium-binding mitochondrial carrier protein SCaMC-1 SLC25A24 APC1 MCSC1 SCAMC1 Mitochondrial ATP-Mg/Pi carrier protein 1 Mitochondrial Ca(2+)-dependent solute carrier protein 1 Small calcium-binding mitochondrial carrier protein 1 Solute carrier family 25 member 24 protein protein human Homo sapiens Human MLRWLRDFVLPTAACQDAEQPTRYETLFQALDRNGDGVVDIGELQEGLRNLGIPLGQDAEEKIFTTGDVNKDGKLDFEEFMKYLKDHEKKMKLAFKSLDKNNDGKIEASEIVQSLQTLGLTISEQQAELILQSIDVDGTMTVDWNEWRDYFLFNPVTDIEEIIRFWKHSTGIDIGDSLTIPDEFTEDEKKSGQWWRQLLAGGIAGAVSRTSTAPLDRLKIMMQVHGSKSDKMNIFGGFRQMVKEGGIRSLWRGNGTNVIKIAPETAVKFWAYEQYKKLLTEEGQKIGTFERFISGSMAGATAQTFIYPMEVMKTRLAVGKTGQYSGIYDCAKKILKHEGLGAFYKGYVPNLLGIIPYAGIDLAVYELLKSYWLDNFAKDSVNPGVMVLLGCGALSSTCGQLASYPLALVRTRMQAQAMLEGSPQLNMVGLFRRIISKEGIPGLYRGITPNFMKVLPAVGISYVVYENMKQTLGVTQK F533D47B2457123F apc4_human Anaphase-promoting complex subunit 4 APC4 Cyclosome subunit 4 ANAPC4 protein protein human Homo sapiens Human MLRFPTCFPSFRVVGEKQLPQEIIFLVWSPKRDLIALANTAGEVLLHRLASFHRVWSFPPNENTGKEVTCLAWRPDGKLLAFALADTKKIVLCDVEKPESLHSFSVEAPVSCMHWMEVTVESSVLTSFYNAEDESNLLLPKLPTLPKNYSNTSKIFSEENSDEIIKLLGDVRLNILVLGGSSGFIELYAYGMFKIARVTGIAGTCLALCLSSDLKSLSVVTEVSTNGASEVSYFQLETNLLYSFLPEVTRMARKFTHISALLQYINLSLTCMCEAWEEILMQMDSRLTKFVQEKNTTTSVQDEFMHLLLWGKASAELQTLLMNQLTVKGLKKLGQSIESSYSSIQKLVISHLQSGSESLLYHLSELKGMASWKQKYEPLGLDAAGIEEAITAVGSFILKANELLQVIDSSMKNFKAFFRWLYVAMLRMTEDHVLPELNKMTQKDITFVAEFLTEHFNEAPDLYNRKGKYFNVERVGQYLKDEDDDLVSPPNTEGNQWYDFLQNSSHLKESPLLFPYYPRKSLHFVKRRMENIIDQCLQKPADVIGKSMNQAICIPLYRDTRSEDSTRRLFKFPFLWNNKTSNLHYLLFTILEDSLYKMCILRRHTDISQSVSNGLIAIKFGSFTYATTEKVRRSIYSCLDAQFYDDETVTVVLKDTVGREGRDRLLVQLPLSLVYNSEDSAEYQFTGTYSTRLDEQCSAIPTRTMHFEKHWRLLESMKAQYVAGNGFRKVSCVLSSNLRHVRVFEMDIDDEWELDESSDEEEEASNKPVKIKEEVLSESEAENQQAGAAALAPEIVIKVEKLDPELDS DIP protein Q9UJX5 original sequence version: 78 DIP protein Q9UJX5 original sequence version: 115 DIP protein Q9UJX5 original sequence version: 120 DIP protein Q9UJX5 original sequence version: 124 DIP protein Q9UJX5 original sequence version: 133 80362CC8D8B2063F apc7_human Anaphase-promoting complex subunit 7 ANAPC7 APC7 Cyclosome subunit 7 protein protein human Homo sapiens Human MDPGDAAILESSLRILYRLFESVLPPLPAALQSRMNVIDHVRDMAAAGLHSNVRLLSSLLLTMSNNNPELFSPPQKYQLLVYHADSLFHDKEYRNAVSKYTMALQQKKALSKTSKVRPSTGNSASTPQSQCLPSEIEVKYKMAECYTMLKQDKDAIAILDGIPSRQRTPKINMMLANLYKKAGQERPSVTSYKEVLRQCPLALDAILGLLSLSVKGAEVASMTMNVIQTVPNLDWLSVWIKAYAFVHTGDNSRAISTICSLEKKSLLRDNVDLLGSLADLYFRAGDNKNSVLKFEQAQMLDPYLIKGMDVYGYLLAREGRLEDVENLGCRLFNISDQHAEPWVVSGCHSFYSKRYSRALYLGAKAIQLNSNSVQALLLKGAALRNMGRVQEAIIHFREAIRLAPCRLDCYEGLIECYLASNSIREAMVMANNVYKTLGANAQTLTLLATVCLEDPVTQEKAKTLLDKALTQRPDYIKAVVKKAELLSREQKYEDGIALLRNALANQSDCVLHRILGDFLVAVNEYQEAMDQYSIALSLDPNDQKSLEGMQKMEKEESPTDATQEEDVDDMEGSGEEGDLEGSDSEAAQWADQEQWFGMQ DIP protein Q9UJX3 original sequence version: 98 DIP protein Q9UJX3 original sequence version: 133 DIP protein Q9UJX3 original sequence version: 143 DIP protein Q9UJX3 original sequence version: 152 D187A1D9DA41DE2C apc1_human Anaphase-promoting complex subunit 1 ANAPC1 TSG24 Cyclosome subunit 1 Testis-specific gene 24 protein Mitotic checkpoint regulator protein protein human Homo sapiens Human MSNFYEERTTMIAARDLQEFVPFGRDHCKHHPNALNLQLRQLQPASELWSSDGAAGLVGSLQEVTIHEKQKESWQLRKGVSEIGEDVDYDEELYVAGNMVIWSKGSKSQALAVYKAFTVDSPVQQALWCDFIISQDKSEKAYSSNEVEKCICILQSSCINMHSIEGKDYIASLPFQVANVWPTKYGLLFERSASSHEVPPGSPREPLPTMFSMLHPLDEITPLVCKSGSLFGSSRVQYVVDHAMKIVFLNTDPSIVMTYDAVQNVHSVWTLRRVKSEEENVVLKFSEQGGTPQNVATSSSLTAHLRSLSKGDSPVTSPFQNYSSIHSQSRSTSSPSLHSRSPSISNMAALSRAHSPALGVHSFSGVQRFNISSHNQSPKRHSISHSPNSNSNGSFLAPETEPIVPELCIDHLWTETITNIREKNSQASKVFITSDLCGQKFLCFLVESQLQLRCVKFQESNDKTQLIFGSVTNIPAKDAAPVEKIDTMLVLEGSGNLVLYTGVVRVGKVFIPGLPAPSLTMSNTMPRPSTPLDGVSTPKPLSKLLGSLDEVVLLSPVPELRDSSKLHDSLYNEDCTFQQLGTYIHSIRDPVHNRVTLELSNGSMVRITIPEIATSELVQTCLQAIKFILPKEIAVQMLVKWYNVHSAPGGPSYHSEWNLFVTCLMNMMGYNTDRLAWTRNFDFEGSLSPVIAPKKARPSETGSDDDWEYLLNSDYHQNVESHLLNRSLCLSPSEASQMKDEDFSQNLSLDSSTLLFTHIPAIFFVLHLVYEELKLNTLMGEGICSLVELLVQLARDLKLGPYVDHYYRDYPTLVRTTGQVCTIDPGQTGFMHHPSFFTSEPPSIYQWVSSCLKGEGMPPYPYLPGICERSRLVVLSIALYILGDESLVSDESSQYLTRITIAPQKLQVEQEENRFSFRHSTSVSSLAERLVVWMTNVGFTLRDLETLPFGIALPIRDAIYHCREQPASDWPEAVCLLIGRQDLSKQACEGNLPKGKSVLSSDVPSGTETEEEDDGMNDMNHEVMSLIWSEDLRVQDVRRLLQSAHPVRVNVVQYPELSDHEFIEEKENRLLQLCQRTMALPVGRGMFTLFSYHPVPTEPLPIPKLNLTGRAPPRNTTVDLNSGNIDVPPNMTSWASFHNGVAAGLKIAPASQIDSAWIVYNKPKHAELANEYAGFLMALGLNGHLTKLATLNIHDYLTKGHEMTSIGLLLGVSAAKLGTMDMSITRLLSIHIPALLPPTSTELDVPHNVQVAAVVGIGLVYQGTAHRHTAEVLLAEIGRPPGPEMEYCTDRESYSLAAGLALGMVCLGHGSNLIGMSDLNVPEQLYQYMVGGHRRFQTGMHREKHKSPSYQIKEGDTINVDVTCPGATLALAMIYLKTNNRSIADWLRAPDTMYLLDFVKPEFLLLRTLARCLILWDDILPNSKWVDSNVPQIIRENSISLSEIELPCSEDLNLETLSQAHVYIIAGACLSLGFRFAGSENLSAFNCLHKFAKDFMTYLSAPNASVTGPHNLETCLSVVLLSLAMVMAGSGNLKVLQLCRFLHMKTGGEMNYGFHLAHHMALGLLFLGGGRYSLSTSNSSIAALLCALYPHFPAHSTDNRYHLQALRHLYVLAAEPRLLVPVDVDTNTPCYALLEVTYKGTQWYEQTKEELMAPTLLPELHLLKQIKVKGPRYWELLIDLSKGTQHLKSILSKDGVLYVKLRAGQLSYKEDPMGWQSLLAQTVANRNSEARAFKPETISAFTSDPALLSFAEYFCKPTVNMGQKQEILDLFSSVLYECVTQETPEMLPAYIAMDQAIRRLGRREMSETSELWQIKLVLEFFSSRSHQERLQNHPKRGLFMNSEFLPVVKCTIDNTLDQWLQVGGDMCVHAYLSGQPLEESQLSMLACFLVYHSVPAPQHLPPIGLEGSTSFAELLFKFKQLKMPVRALLRLAPLLLGNPQPMVM DIP protein Q9H1A4 original sequence version: 85 DIP protein Q9H1A4 original sequence version: 119 DIP protein Q9H1A4 original sequence version: 128 DIP protein Q9H1A4 original sequence version: 137 E08B1FE3FC28917E nek2_human Serine/threonine-protein kinase Nek2 NEK2A NimA-like protein kinase 1 HSPK 21 NEK2 NLK1 Never in mitosis A-related kinase 2 protein protein human Homo sapiens Human MPSRAEDYEVLYTIGTGSYGRCQKIRRKSDGKILVWKELDYGSMTEAEKQMLVSEVNLLRELKHPNIVRYYDRIIDRTNTTLYIVMEYCEGGDLASVITKGTKERQYLDEEFVLRVMTQLTLALKECHRRSDGGHTVLHRDLKPANVFLDGKQNVKLGDFGLARILNHDTSFAKTFVGTPYYMSPEQMNRMSYNEKSDIWSLGCLLYELCALMPPFTAFSQKELAGKIREGKFRRIPYRYSDELNEIITRMLNLKDYHRPSVEEILENPLIADLVADEQRRNLERRGRQLGEPEKSQDSSPVLSELKLKEIQLQERERALKAREERLEQKEQELCVRERLAEDKLARAENLLKNYSLLKERKFLSLASNPELLNLPSSVIKKKVHFSGESKENIMRSENSESQLTSKSKCKDLKKRLHAAQLRAQALSDIEKNYQLKSRQILGMR D33A37778ABB6D9E bub1b_human Mitotic checkpoint serine/threonine-protein kinase BUB1 beta MAD3/BUB1-related protein kinase Mitotic checkpoint kinase MAD3L SSK1 BUB1B BUBR1 MAD3L Protein SSK1 protein protein human Homo sapiens Human MAAVKKEGGALSEAMSLEGDEWELSKENVQPLRQGRIMSTLQGALAQESACNNTLQQQKRAFEYEIRFYTGNDPLDVWDRYISWTEQNYPQGGKESNMSTLLERAVEALQGEKRYYSDPRFLNLWLKLGRLCNEPLDMYSYLHNQGIGVSLAQFYISWAEEYEARENFRKADAIFQEGIQQKAEPLERLQSQHRQFQARVSRQTLLALEKEEEEEVFESSVPQRSTLAELKSKGKKTARAPIIRVGGALKAPSQNRGLQNPFPQQMQNNSRITVFDENADEASTAELSKPTVQPWIAPPMPRAKENELQAGPWNTGRSLEHRPRGNTASLIAVPAVLPSFTPYVEETARQPVMTPCKIEPSINHILSTRKPGKEEGDPLQRVQSHQQASEEKKEKMMYCKEKIYAGVGEFSFEEIRAEVFRKKLKEQREAELLTSAEKRAEMQKQIEEMEKKLKEIQTTQQERTGDQQEETMPTKETTKLQIASESQKIPGMTLSSSVCQVNCCARETSLAENIWQEQPHSKGPSVPFSIFDEFLLSEKKNKSPPADPPRVLAQRRPLAVLKTSESITSNEDVSPDVCDEFTGIEPLSEDAIITGFRNVTICPNPEDTCDFARAARFVSTPFHEIMSLKDLPSDPERLLPEEDLDVKTSEDQQTACGTIYSQTLSIKKLSPIIEDSREATHSSGFSGSSASVASTSSIKCLQIPEKLELTNETSENPTQSPWCSQYRRQLLKSLPELSASAELCIEDRPMPKLEIEKEIELGNEDYCIKREYLICEDYKLFWVAPRNSAELTVIKVSSQPVPWDFYINLKLKERLNEDFDHFCSCYQYQDGCIVWHQYINCFTLQDLLQHSEYITHEITVLIIYNLLTIVEMLHKAEIVHGDLSPRCLILRNRIHDPYDCNKNNQALKIVDFSYSVDLRVQLDVFTLSGFRTVQILEGQKILANCSSPYQVDLFGIADLAHLLLFKEHLQVFWDGSFWKLSQNISELKDGELWNKFFVRILNANDEATVSVLGELAAEMNGVFDTTFQSHLNKALWKVGKLTSPGALLFQ DIP protein O60566 original sequence version: 106 DIP protein O60566 original sequence version: 130 DIP protein O60566-1 original sequence version: 119 DIP protein O60566-1 original sequence version: 154 DIP protein O60566 original sequence version: 154 F7871103A56E6B46 bub3_human Mitotic checkpoint protein BUB3 BUB3 protein protein human Homo sapiens Human MTGSNEFKLNQPPEDGISSVKFSPNTSQFLLVSSWDTSVRLYDVPANSMRLKYQHTGAVLDCAFYDPTHAWSGGLDHQLKMHDLNTDQENLVGTHDAPIRCVEYCPEVNVMVTGSWDQTVKLWDPRTPCNAGTFSQPEKVYTLSVSGDRLIVGTAGRRVLVWDLRNMGYVQQRRESSLKYQTRCIRAFPNKQGYVLSSIEGRVAVEYLDPSPEVQKKKYAFKCHRLKENNIEQIYPVNAISFHNIHNTFATGGSDGFVNIWDPFNKKRLCQFHRYPTSIASLAFSNDGTTLAIASSYMYEMDDTEHPEDGIFIRQVTDAETKPKSPCT DIP protein O43684 original sequence version: 108 DIP protein O43684 original sequence version: 140 2915572A57368E5A ki18a_human Kinesin-like protein KIF18A KIF18A OK/SW-cl.108 Marrow stromal KIF18A protein protein human Homo sapiens Human MSVTEEDLCHHMKVVVRVRPENTKEKAAGFHKVVHVVDKHILVFDPKQEEVSFFHGKKTTNQNVIKKQNKDLKFVFDAVFDETSTQSEVFEHTTKPILRSFLNGYNCTVLAYGATGAGKTHTMLGSADEPGVMYLTMLHLYKCMDEIKEEKICSTAVSYLEVYNEQIRDLLVNSGPLAVREDTQKGVVVHGLTLHQPKSSEEILHLLDNGNKNRTQHPTDMNATSSRSHAVFQIYLRQQDKTASINQNVRIAKMSLIDLAGSERASTSGAKGTRFVEGTNINRSLLALGNVINALADSKRKNQHIPYRNSKLTRLLKDSLGGNCQTIMIAAVSPSSVFYDDTYNTLKYANRAKDIKSSLKSNVLNVNNHITQYVKICNEQKAEILLLKEKLKAYEEQKAFTNENDQAKLMISNPQEKEIERFQEILNCLFQNREEIRQEYLKLEMLLKENELKSFYQQQCHKQIEMMCSEDKVEKATGKRDHRLAMLKTRRSYLEKRREEELKQFDENTNWLHRVEKEMGLLSQNGHIPKELKKDLHCHHLHLQNKDLKAQIRHMMDLACLQEQQHRQTEAVLNALLPTLRKQYCTLKEAGLSNAAFESDFKEIEHLVERKKVVVWADQTAEQPKQNDLPGISVLMTFPQLGPVQPIPCCSSSGGTNLVKIPTEKRTRRKLMPSPLKGQHTLKSPPSQSVQLNDSLSKELQPIVYTPEDCRKAFQNPSTVTLMKPSSFTTSFQAISSNINSDNCLKMLCEVAIPHNRRKECGQEDLDSTFTICEDIKSSKCKLPEQESLPNDNKDILQRLDPSSFSTKHSMPVPSMVPSYMAMTTAAKRKRKLTSSTSNSSLTADVNSGFAKRVRQDNSSEKHLQENKPTMEHKRNICKINPSMVRKFGRNISKGNLR 6DFD904378BF9B27 anapc4-kif18a 3 KIF18A 13 ms participant Identification by mass spectrometry ms participant experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level NEK2A 10 ms participant Identification by mass spectrometry ms participant experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f1a homomint mint The APC/C was purified from nocodazole-arrested cells and cells that were released from nocodazole into medium containing the proteasome inhibitor MG132 (Figure 1A). These, and control preparations, were analysed by mass spectrometry. In APC/C purifications from both conditions we identified two peptides of the kinesin Kif18A and in the sample from cells in metaphase we identified 14 peptides of Nek2A. anapc4-nek2 3 APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level NEK2A 10 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell P51955:p.Leu320Ala mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L A Tue Jul 12 10:19:16 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 17:29:53 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu320ala' Tue Jun 30 20:05:31 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu320Ala' binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain leucin zipper tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined P51955:p.Leu313Ala mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L A Tue Jul 12 10:56:07 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 14:31:52 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu313ala' Tue Jun 30 15:53:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu313Ala' undefined mutation mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain Wed Jul 06 17:43:19 BST 2016 Sequence change details about this feature cannot be ascertained or do not fit with the current version of the referenced protein, so they have been deleted as a result of our quality control procedures. The original label was 'mutation decreasing' 325-340 physical association physical association homomint mint f5a f5b f5c f5d f5e To understand why the leucine zipper was required for Nek2A degradation during an active checkpoint, we analysed the ability of the different Nek2A constructs to bind to the APC/C. Nek2A and Kif18A constructs were transfected into HeLa cells and the APC/C purified from nocodazole-arrested cells (Figure 5A C). From these experiments, it was clear that both the C-terminal MR motif and leucine zipper of Nek2A were required for high affinity binding to the APC/C, even though the leucine zipper alone did not bind to the APC/C (Figure 5B). As the leucine zipper mediates dimerization of Nek2A we wanted to address whether Nek2A had to be a dimer to bind with high affinity to the APC/C. We mutated two critical leucine residues in the leucine zipper predicted to be required for dimerization (Croasdale et al, 2011), L313A/L320A, and analysed the ability of this mutant to bind the APC/C. At high expression levels, there was still detectable binding of Nek2A L313A/L320A to the APC/C although it was reduced compared to wild type (Figure 5B and C). However, at lower levels of expression Nek2A L313A/L320A was strongly impaired in binding to the APC/C (Figure 5D and E). anapc4-kif18a-1 3 APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level KIF18A 13 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell flag tag 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 f1c homomint mint anapc4-nek2-1 3 APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level NEK2A 10 ms participant Identification by mass spectrometry ms participant experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f1a homomint mint The APC/C was purified from nocodazole-arrested cells and cells that were released from nocodazole into medium containing the proteasome inhibitor MG132 (Figure 1A). These, and control preparations, were analysed by mass spectrometry. In APC/C purifications from both conditions we identified two peptides of the kinesin Kif18A and in the sample from cells in metaphase we identified 14 peptides of Nek2A. anapc4-bub3 3 Cdc20 4 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level NEK2A 10 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain flag tag 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 MAD2 5 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Bub3 12 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association homomint mint sf4b To determine whether Nek2A was able to displace the MCC from the APC/C, we overexpressed Nek2A more than a 100-fold above its endogenous levels and analysed MCC binding to the APC/C. We only observed a slight decrease in MCC binding upon overexpression of Nek2A in two out of five experiments, indicating that competition with the MCC was an unlikely mechanism by which Nek2A escapes SAC inhibition (Supplementary Figure S4B). anapc4-anapc7 3 BubR1 11 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC7 8 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC1 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level NEK2A 10 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f6a homomint mint bub1b-anapc4-1 3 BubR1 11 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC7 8 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC1 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f6a homomint mint nek2-anapc1 2 NEK2A 10 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined APC1 9 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level NEK2A 10 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined P51955:p.Leu320Ala mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L A Tue Jul 12 10:23:54 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 16:46:30 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu320ala' Tue Jun 30 16:16:59 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu320Ala' binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain leucin zipper P51955:p.Leu313Ala mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L A Tue Jul 12 10:58:58 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 15:22:54 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu313ala' Tue Jun 30 18:34:48 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu313Ala' APC7 8 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association homomint mint f5f f5g We also performed purifications of Nek2A, Nek2A L313A/L320A and Nek2A D310 325 and analysed the binding both to APC/C and to myc-tagged Nek2A. Again Nek2A L313A/L320A and Nek2A D310 325 were strongly impaired in binding to the APC/C and this correlated exactly with the level of binding to myc Nek2A (Figure 5F and G). anapc4-anapc7-1 1 BubR1 11 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level MAD2 5 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level NEK2A 10 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC4 7 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level APC7 8 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f6b homomint mint