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/ LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. J. Biol. Chem. (0021-9258) 2004 imex curation Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions mouse-myeloma Mus musculus myeloma cell line myeloma lymphoblast plasma cell line. coip coimmunoprecipitation co-immunoprecipitation CoIp immunoprecipitation Co-IP co-immunoprecipitation CoIp immunoprecipitation Co-IP coip western blot western blot Immuno blot Immuno blot Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu Accepted 2009-APR-31 by DAVET. 09-April-2009: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 davet Only protein-protein interactions imex curation LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. J. Biol. Chem. (0021-9258) 2004 imex curation Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 coip coimmunoprecipitation co-immunoprecipitation CoIp immunoprecipitation Co-IP co-immunoprecipitation CoIp immunoprecipitation Co-IP coip western blot western blot Immuno blot Immuno blot Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu Accepted 2009-APR-31 by DAVET. 09-April-2009: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 davet Only protein-protein interactions imex curation LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. J. Biol. Chem. (0021-9258) 2004 imex curation Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions chlae-ts_cos Chlorocebus aethiops Grivet ts_cos Simian cells (cercopithecus aethiops) transformed by SV40 pull down pull down anti tag western anti tag western blot anti tag western Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu Accepted 2009-APR-31 by DAVET. 09-April-2009: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 davet Only protein-protein interactions imex curation LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. LEK1 is a potential inhibitor of pocket protein-mediated cellular processes. J. Biol. Chem. (0021-9258) 2004 imex curation Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Ashe M., Pabon-Pena L., Dees E., Price KL., Bader D. david.bader@mcmail.vanderbilt.edu Accepted 2009-APR-31 by DAVET. 09-April-2009: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 davet Only protein-protein interactions imex curation q155p7_mouse Cenpf Lek1 protein protein mouse Mus musculus Mouse MSWALEEWKEGLPSRALQKIQELEGQLEKLKKEKQQRQFQLDSLEAALQKQKQKVEDGKTEGADLKRENQRLMEICEHLEKSRQKLSHELQVKESQVNLQESQLSSCKKQIEKLEQELKRCKSEFERSQQVAQSADVSLNPCSTPQKLFATPLTPSSTYEDLKEKYNKEVEERKRLEEEVKALHAKKVSLPVSQATMNHRDIARHQASSSVFPWQQENTPSRLSSDALKTPLRRDGSAAHFLGEEVSPNKSSMKTGRGDCSSLPGEPHSAQLLHQAKAQNQDLKSKMTELELRLQGQEKEMRSQVNKCQDLQLQLEKTKVELIEKERILNKTRDEVVRSTAQYDQAAAKCTTLEQKLKTLTEELSCHRQNAESAKRSLEQRIKEKEKELQEELSRQHQSFQALDSEYTQMKTRLTQELQQVKHLHSTLQLELEKVTSVKQQLERNLEEIRLKLSRAEQALQASQVAENELRRSSEEMKKENSLIRSQSEQRTREVCHLEEELGKVKVSLSKSQNFAEEMKAKNTSQEIMLRDLQEKLNQQENSLTLEKLKLALADLERQRNCSQDLLKKREHHIDQLNNKLNKIEKEFETLLSALELKKKECEELKEEKNQISFWKIDSEKLINQIESEKEILLGKINHLETSLKTQQVSPDSNERIRTLEMERENFTVEIKNLQSMLDSKMVEIKTQKQAYLELQQKSESSDQKHQKEIENMCLKANKLTGQVESLECKLQLLSSEVVTKDQQYQDLRMEYETLRDLLKSRGSSLVTNEDNQRSSEDNQRSSEDNQRGSLAFEQQPAVSDSFANVMGRKGSINSERSDCSVDGGRSPEHIAILQNRVTSLESSLESQNQMNSDLQMRCEELLQIKGEVEENLSKAEQIHQNFVAETNQCISKLQEDAAVHQNIVAETLATLESKEKELQLLKEKLEAQQTEVQKLNKNNCLLEGTLKELQLLSDTLSSEKKEMNSIISLSKKNIEELTQANEALKEVNEALEQEKMNLLQKHEKITSCIAEQERSIAELSDQYKQERLQLLQRCEETEAVLEDLRGNYKTAQENNAKLECMLSECTALCENRKNELEQLKETFAKEQQEFLTKLAFAEEQNRKLMLELEIEQQTVRSEITNTNKHSMSATDGLRQECLTLNEEQNEQQNEVSNLTHENEQLMELTQTKHDSYLAVEPVENSVKATEDEIGKSSSQYQMDIDTKDISLDSYKAQLVHLEALVRILEVQLDQSEEENKKLHLELQTIREELETKSSQDPQSQARTGLKDCDTAEEKYVSMLQELSASQNENAHLQCSLQTAVNKLNELGKMCDVLRVEKLQLESELNDSRTECITATSQMTAEVEKLVSEMKMLNHESALSQNELMKDTSGGEFHDKANHSSVFLTPLDSSNFCEQMTLSSKEVRVHFAELQEKFSCLQSEHKILHDQHCEVSSKMSALRSYVDTLKAENSALSMSLRTLQGDLVKEGEPAAEGGHGLPLSFCGADSPSLTNFGETSFYKDVLEQTGDTCHLSLEGNASANSCDLDEEFSSSLEEETLTEKESPPAPGRTVEGLEVLCQVYLQSLKNLEEKTESQRIMKNKEIEELEQLLSSERKELSCLRKQYLSEKEQWQQKLTSVTLEMESKLAEEKQQTKTLSLELEVARLQLQELDLSSRSLLGTDLESVVRCQNDNYDIKESEVYISETTEKTPKQDTDQTCDKDIQQDLGLETSVTESETTRLTGEGCEEQPPKTNCEAPAEDKTQDCSECISELCSSSNVLVPMDVLEDQGSIQNLQLQKDTLNENLRLLPEVEDWDKKVESLLNEIMEADSKLSLQEVQLKMKIATCIQLEKIVKDLRKEKADLSEKLESLPCNQEVCLRVERSEEDLGFNLDMGANELLSKSTKDNATNTEDNYKEKFLDMERELTRIKSEKANIEHHILSVETNLEVVQAEKLCLERDTESKQKVIIDLKEELFTVISERNRLREELDNVSKESKALDQMSKKMKEKIEELESHQRESLRHIGAVESEVKDKADLIQTLSFNVGELTKDKAHLQEQLQNLQNDSQELSLAIGELEIQIGQLNKEKESLVKESQNFQIKLTESECEKQTISKALEVALKEKGEFAVQLSSAQEEVHQLRRGIEKLSVRIEADEKKHLSAVAKLKESQRESDSLKDTVETLERELERSEENQELAILDSENLKAEVETLKAQKDEMTKSLRIFELDLVTVRTERENLAKQLQEKQSRVSELDERCSSLRRLLEEKEQARVQMEEDSKSAMLMLQMQLKELREEVAALCNDQETLKAQEQSLDQPGEEVHHLKSSIRKLKVHIDADEKKHQNILEQLKESKHHADLLKDRVENLEQELILSEKNMIFQAEKSKAEIQTLKSEIQRMAQNLQDLQLELISTRSENENLIKELKKEQERVSDLETINPSIENLLKDKEQEKVQMKEEAKITVEMLQTQLKELNETVVSLCNDQEVSKTKEQNLGSQVQTLELEKAQLLQDLGEAKNKYIIFQSSVNALTQEVEAGKQKLEKGEKEIRTLKEQLKSQEQLVCKLAQVEGEQELWQKQKLELRNVTMALEQKVQVLQSENNTLQSTYEALQNSHKSLESELGLIKLEKVALVERVSTISGKEAELQRELRDMLQKTTQLSEDYNKEKNRLTEEVEVLREELQNTKAAHLKSVNQLEKELQRAQGKIKLMLKSCRQLEGEKEMLQKELSQLEAAQQQRAGSLVDSNVDEVMTENKALKETLEEKVKEADKYLDKYCSLLISHEELEKAKEILEIEVARLKSRQSRQDLQSSPLLNSSIPGPSPNTSVSEMKSASGQNKASGKRQRSSGVWEHGKRAAPSTAETFSKKSRKSDSKSTRPAEHEQETEFEPEGLPEVVKKGFADIPTGKTSPYILRRTTMATRTSPRFATQKLVGSSPSLGKENVVESSKPTAGGSRSQKVKVVQESSADSHTAFQELPAKSLTASNIPGRNSTESPREGLRAKRAYPASSPAAGPDPTNNENCRVQ 3CB74C4F24537155 rb_human Retinoblastoma-associated protein pRb pp110 p105-Rb RB1 p110-RB1 protein protein human Homo sapiens Human MPPKTPRKTAATAAAAAAEPPAPPPPPPPEEDPEQDSGPEDLPLVRLEFEETEEPDFTALCQKLKIPDHVRERAWLTWEKVSSVDGVLGGYIQKKKELWGICIFIAAVDLDEMSFTFTELQKNIEISVHKFFNLLKEIDTSTKVDNAMSRLLKKYDVLFALFSKLERTCELIYLTQPSSSISTEINSALVLKVSWITFLLAKGEVLQMEDDLVISFQLMLCVLDYFIKLSPPMLLKEPYKTAVIPINGSPRTPRRGQNRSARIAKQLENDTRIIEVLCKEHECNIDEVKNVYFKNFIPFMNSLGLVTSNGLPEVENLSKRYEEIYLKNKDLDARLFLDHDKTLQTDSIDSFETQRTPRKSNLDEEVNVIPPHTPVRTVMNTIQQLMMILNSASDQPSENLISYFNNCTVNPKESILKRVKDIGYIFKEKFAKAVGQGCVEIGSQRYKLGVRLYYRVMESMLKSEEERLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTSQNLDSGTDLSFPWILNVLNLKAFDFYKVIESFIKAEGNLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDREGPTDHLESACPLNLPLQNNHTAADMYLSPVRSPKKKGSTTRVNSTANAETQATSAFQTQKPLKSTSLSLFYKKVYRLAYLRLNTLCERLLSEHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAVQETFKRVLIKEEEYDSIIVFYNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFPSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRILVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGSNPPKPLKKLRFDIEGSDEADGSKHLPGESKFQQKLAEMTSTRTRMQKQKMNDSMDTSNKEEK DIP protein P06400 original sequence version: 102 DIP protein P06400 original sequence version: 195 DIP protein P06400 original sequence version: 204 DIP protein P06400 original sequence version: 126 DIP protein P06400 original sequence version: 151 DIP protein P06400 original sequence version: 153 DIP protein P06400 original sequence version: 181 DIP protein P06400 original sequence version: 184 DIP protein P06400 original sequence version: 213 DIP protein P06400 original sequence version: 208 DIP protein P06400 original sequence version: 174 DIP protein P06400 original sequence version: 212 DIP protein P06400 original sequence version: 179 DIP protein P06400 original sequence version: 146 C8E746111E19CC32 rbl1_human Retinoblastoma-like protein 1 RBL1 pRb1 107 kDa retinoblastoma-associated protein protein protein human Homo sapiens Human MFEDKPHAEGAAVVAAAGEALQALCQELNLDEGSAAEALDDFTAIRGNYSLEGEVTHWLACSLYVACRKSIIPTVGKGIMEGNCVSLTRILRSAKLSLIQFFSKMKKWMDMSNLPQEFRERIERLERNFEVSTVIFKKYEPIFLDIFQNPYEEPPKLPRSRKQRRIPCSVKDLFNFCWTLFVYTKGNFRMIGDDLVNSYHLLLCCLDLIFANAIMCPNRQDLLNPSFKGLPSDFHTADFTASEEPPCIIAVLCELHDGLLVEAKGIKEHYFKPYISKLFDRKILKGECLLDLSSFTDNSKAVNKEYEEYVLTVGDFDERIFLGADAEEEIGTPRKFTRDTPLGKLTAQANVEYNLQQHFEKKRSFAPSTPLTGRRYLREKEAVITPVASATQSVSRLQSIVAGLKNAPSDQLINIFESCVRNPVENIMKILKGIGETFCQHYTQSTDEQPGSHIDFAVNRLKLAEILYYKILETVMVQETRRLHGMDMSVLLEQDIFHRSLMACCLEIVLFAYSSPRTFPWIIEVLNLQPFYFYKVIEVVIRSEEGLSRDMVKHLNSIEEQILESLAWSHDSALWEALQVSANKVPTCEEVIFPNNFETGNGGNVQGHLPLMPMSPLMHPRVKEVRTDSGSLRRDMQPLSPISVHERYSSPTAGSAKRRLFGEDPPKEMLMDKIITEGTKLKIAPSSSITAENVSILPGQTLLTMATAPVTGTTGHKVTIPLHGVANDAGEITLIPLSMNTNQESKVKSPVSLTAHSLIGASPKQTNLTKAQEVHSTGINRPKRTGSLALFYRKVYHLASVRLRDLCLKLDVSNELRRKIWTCFEFTLVHCPDLMKDRHLDQLLLCAFYIMAKVTKEERTFQEIMKSYRNQPQANSHVYRSVLLKSIPREVVAYNKNINDDFEMIDCDLEDATKTPDCSSGPVKEERGDLIKFYNTIYVGRVKSFALKYDLANQDHMMDAPPLSPFPHIKQQPGSPRRISQQHSIYISPHKNGSGLTPRSALLYKFNGSPSKSLKDINNMIRQGEQRTKKRVIAIDSDAESPAKRVCQENDDVLLKRLQDVVSERANH DIP protein P28749 original sequence version: 154 2BC4876EA04BDB31 rbl1_mouse Retinoblastoma-like protein 1 Rbl1 pRb1 107 kDa retinoblastoma-associated protein protein protein mouse Mus musculus Mouse MFEDEPHAEGAAAVAAAREALQALCQELNLDEGSAAEALDDFTAIRGNYSLEGEVIHWLACSLYVACRKSIIPTVGKGVMEGNCVSLTRILRSAKLSLIQFFSKMKKWMDMSNLPQEFRERIERLERNFEVSTVIFKKFEPIFLDIFQNPYEEPPKLPRSRKQRRIPCSVKDLFNFCWTLFVYTKGNFRMIGDDLVNSYHLLLCCLDLIFANAIMCPNRRDLLNPSFKGLPSDFHAPDFKAAEEPPCIIAVLCDLHDGLLVEAKGIKEHYFKPYISKLFDKKILKGECLLDLSSFTDNSKAVNKEYEEYVLTVGDFDERIFLGADAEEEIGTPRKFTADTPFGKLTSQASVECNLQQHFEKKRSFAPSTPLTGRRYLQEKEAVTTPVASATQSVSRLQSIVAGLKSAPSEQLLNIFESCMRNPMGNIIKIVKGIGETFCQHYTQSTDKQPGSHIDFAVNRLKLAEILYYKILETIMVQETRRLHGMDMSVLLEQDIFHKSLMACCLEIVLFAYSSPRTFPWIIEVLDLQPFYFYKVIEVVIRSEEGLSRDMVKHLNSIEEQILESLAWTNNSALWEALHASANRVPSCEEVIFPNNFEIGNGGNVQGHLPMMPMSPIIHPRVKEVRTDSGSLRQDMQPLSPISVHERYSSPAAGSAKRRLFGDDPPKDTLMDKIMAEGTKLKIAPSSVTAESLSISPGQALLTMATTTVTGTTGRKVTVPLHGIANDAGEITLVPISMNPTQESTAESPVSLTAQSLIGTSPKQTHLTKAQDAHLTGVSKPKRTGSLALFYRKVYHLASVRLRDLCLKLDVSNELRRKIWTCFEFTLVHCPDLMKDRHLDQLLLCAFYIMAKVTKEERTFQEIMKSYRNQPQANSHVYRSVLLKSIPGGVVVYNGDCEMTDGDIEDATKTPNCSSEPVKEERGDLIKFYNTVYVGRVKSFALKYDLSNQDHIMDAPPLSPFPHIKQQPGSPRRISQQHSLYVSPHKNGAGLTPRSALLYKFNGSPSKSLKDINNMIRQGEQKTKKRVIAISGDADSPAKRLCQENDDVLLKRLQDVVSERANH 85BEA8EC064EC06E rbl2_mouse Retinoblastoma-like protein 2 Rbl2 Retinoblastoma-related protein 2 130 kDa retinoblastoma-associated protein pRb2 protein protein mouse Mus musculus Mouse MASGGNQSPPPPPAAAASSEEEEEDGDAADRAQPAGSPSHQIQQRFEELCSRLNMDEAARAEAWSSYRSMSESYTLEGNDLHWLACALYVACRKSVPTVSKGTAEGNYVSLTRILRCSEQSLIEFFNKMKKWEDMANLPPHFRERTERLERNFTVSAVIFKKYEPIFQDIFKYPQEEQPRQQRGRKQRRQPCTTSEIFHFCWVLFIYAKGNFPMISDDLVNSYHLLLCALDLVYGNALQCSNRKELVNPNFKGLSEDCHPKDSKASSDPPCVIEKLCSLHDGLVLEAKGIKEHFWKPYIRKLFEKKLLKGKEENLTGFLEPGNFGESFKAVNKAYEEYVLAAGNLDERVFLGEDAEEEVGTLSRCLSAASGTESAERTQMRDILQQHLDKSKALRVCTPLTGVRYVQENSPCVTPVSTAAHSLSRLHTMLSGLRNAPSEKLERILRSCSRDPTQAIADRLKEMYEIYSQHFQPDENFSNCAKEIANKHFRFAEMLYYKVLESVIEQEQKRLGDMDLSGVLEHDAFHRSLLACCLEVVAFSHKPPGNFPFIAEIFDVPHYHFYKVIEVFIRAEDGLCREVVKHLNQIEEQILDHLAWKTKSPLWDRIRDNENRVPTCEEVMPPQNLERTDEIYIAGSPLTPRRVGEVRADAGGLGRSITSPTTLYDRYSSPTVSTTRRRLFENDSPSEGSTSGRIPPQPLVNAVPVQNVPGETVSVTPVPGQTLVTMATATVTANNGQTVTIPVQGIANENGGITFFPVQVNVGGQAQAVAGSIQPLSAQALAGSLSSQQVTGTTLQVPGPVAIQQISPGGQQQNPGQPLTSSSIRPRKTSSLALFFRKVYYLAGVRLRDLCIKLDISDELRKKIWTCFEFSIIQCTELMMDRHLDQLLMCAIYVMAKVTKEDRSFQNIMRCYRTQPQARSQVYRSVLIKGKRRNSGSSESRSHQNSPTELNTDRASRDSSPVMRSNSTLPVPQPSSAPPTPTRLTGASSDVEEEERGDLIQFYNNIYRKQIQAFAMKYSQANAQTDTPPLSPYPFVRTGSPRRVQLSQSHPIYISPHNNEAMPSPREKIFYYFSNSPSKRLREINSMIRTGETPTKKRGILLDDGSESPAKRICPENHSALLRRLQDVANDRGSQ F509E68A156929B2 rb_mouse Retinoblastoma-associated protein Rb1 Rb-1 pRb pp105 p110-RB1 protein protein mouse Mus musculus Mouse MPPKAPRRAAAAEPPPPPPPPPREDDPAQDSGPEELPLARLEFEEIEEPEFIALCQKLKVPDHVRERAWLTWEKVSSVDGILEGYIQKKKELWGICIFIAAVDLDEMPFTFTELQKSIETSVYKFFDLLKEIDTSTKVDNAMSRLLKKYNVLCALYSKLERTCELIYLTQPSSALSTEINSMLVLKISWITFLLAKGEVLQMEDDLVISFQLMLCVVDYFIKFSPPALLREPYKTAAIPINGSPRTPRRGQNRSARIAKQLENDTRIIEVLCKEHECNIDEVKNVYFKNFIPFINSLGIVSSNGLPEVESLSKRYEEVYLKNKDLDARLFLDHDKTLQTDPIDSFETERTPRKNNPDEEANVVTPHTPVRTVMNTIQQLMVILNSASDQPSENLISYFNNCTVNPKENILKRVKDVGHIFKEKFANAVGQGCVDIGVQRYKLGVRLYYRVMESMLKSEEERLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTLQHLDSGTDLSFPWILNVLNLKAFDFYKVIESFIKVEANLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDGEGPDNLEPACPLSLPLQGNHTAADMYLSPLRSPKKRTSTTRVNSAANTETQAASAFHTQKPLKSTSLALFYKKVYRLAYLRLNTLCARLLSDHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAAQETFKRVLIREEEFDSIIVFYNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFSSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRILVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGGNPPKPLKKLRFDIEGADEADGSKHLPAESKFQQKLAEMTSTRTRMQKQRMNESKDVSNKEEK DIP protein P13405 original sequence version: 115 DIP protein P13405 original sequence version: 114 DIP protein P13405 original sequence version: 110 AE81D35A07ADB493 cenpf-rb1 4 10 unspecified role unspecified role prey prey spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 ecoli Escherichia coli (strain K12) physical association physical association Fig 2. Fig. 2 demonstrates that COOH-terminal LEK1 forms complexes with hypophosphorylated full-length pRb, as is observed by the presence of hypophosphorylated pRb in the LEK1 precipitate at increasing concentrations. pRb did not interact with GST alone. The C-terminal portion of LEK1 interacts with hypophosphorylated full-length Rb1 The authors have not given the exact positions of the c-terminus they have used in their interaction studies. I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. The authors do not state the exact insect cells used in the generation of wt Rb using the Baculoviral expression system but doing a google and pubmed search, the most common insects cells for this are Sf9 cells and so that is what I have stated that the Rb protein is expressed in. cenpf-rb1-1 2 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis c-term range C-terminal range c-term range c-term range C-terminal range c-term range region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey physical association physical association Fig 3A and 3B. Constituents of these immune complexes were assayed by Western blot (Fig. 3). Our results show that all three members of the Rb family are able to form complexes with LEK1 (Fig. 3A). Experiments using anti-LEK1 antisera to perform immunoprecipitations yielded the same results, demonstrating that all three Rb proteins coprecipitate with LEK1 (Fig. 3B). LEK1 associates with not just one, but all three members of the Rb family, suggesting that LEK1 has the potential to affect the activity of all members of the Rb family. The authors have not given the exact positions of the c-terminus they have used in their interaction studies. I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rbl1 2 8 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis c-term range C-terminal range c-term range c-term range C-terminal range c-term range region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig 3A and 3B. Constituents of these immune complexes were assayed by Western blot (Fig. 3). Our results show that all three members of the Rb family are able to form complexes with LEK1 (Fig. 3A). Experiments using anti-LEK1 antisera to perform immunoprecipitations yielded the same results, demonstrating that all three Rb proteins coprecipitate with LEK1 (Fig. 3B). LEK1 associates with not just one, but all three members of the Rb family, suggesting that LEK1 has the potential to affect the activity of all members of the Rb family. The authors have not given the exact positions of the c-terminus they have used in their interaction studies. I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rbl2 2 9 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis c-term range C-terminal range c-term range c-term range C-terminal range c-term range region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig 3A and 3B. Constituents of these immune complexes were assayed by Western blot (Fig. 3). Our results show that all three members of the Rb family are able to form complexes with LEK1 (Fig. 3A). Experiments using anti-LEK1 antisera to perform immunoprecipitations yielded the same results, demonstrating that all three Rb proteins coprecipitate with LEK1 (Fig. 3B). LEK1 associates with not just one, but all three members of the Rb family, suggesting that LEK1 has the potential to affect the activity of all members of the Rb family. The authors have not given the exact positions of the c-terminus they have used in their interaction studies. I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rb1-2 3 6 unspecified role unspecified role prey prey c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 Fig 5A and 5B. GST-Rb fusion proteins were conjugated to glutathione-Sepharose beads and coincubated with whole cell lysates of COS cells that had been transiently transfected with LEK1 expression plasmid (Fig. 5A). GST-Rb-(379�928), which contains the A, B, and C pockets (the long pocket), binds to LEK1. GST-Rb-(379�792), which only contains the A/B pocket region, also interacts at similar levels with LEK1. Furthermore, GST-Rb-(792�928), the fusion protein that only contains the C pocket, was also able to interact with LEK1 (Fig. 5B). This suggests that LEK1 is able to recognize sequences in all three pocket subdomains of Rb. The C-terminal portion of LEK1 interacts with sequences in all three pocket subdomains of Rb. I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rb1-3 3 6 unspecified role unspecified role prey prey 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 c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 Fig 5A and 5B. GST-Rb fusion proteins were conjugated to glutathione-Sepharose beads and coincubated with whole cell lysates of COS cells that had been transiently transfected with LEK1 expression plasmid (Fig. 5A). GST-Rb-(379�928), which contains the A, B, and C pockets (the long pocket), binds to LEK1. GST-Rb-(379�792), which only contains the A/B pocket region, also interacts at similar levels with LEK1. Furthermore, GST-Rb-(792�928), the fusion protein that only contains the C pocket, was also able to interact with LEK1 (Fig. 5B). This suggests that LEK1 is able to recognize sequences in all three pocket subdomains of Rb. The C-terminal portion of LEK1 interacts with sequences in all three pocket subdomains of Rb. I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rb1-4 3 6 unspecified role unspecified role prey prey 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 c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 Fig 5A and 5B. GST-Rb fusion proteins were conjugated to glutathione-Sepharose beads and coincubated with whole cell lysates of COS cells that had been transiently transfected with LEK1 expression plasmid (Fig. 5A). GST-Rb-(379�928), which contains the A, B, and C pockets (the long pocket), binds to LEK1. GST-Rb-(379�792), which only contains the A/B pocket region, also interacts at similar levels with LEK1. Furthermore, GST-Rb-(792�928), the fusion protein that only contains the C pocket, was also able to interact with LEK1 (Fig. 5B). This suggests that LEK1 is able to recognize sequences in all three pocket subdomains of Rb. The C-terminal portion of LEK1 interacts with sequences in all three pocket subdomains of Rb. I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rb1-5 3 5 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 c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey cys-709-phe binding region binding-associated region binding region mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain 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 c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association Fig 5C. To determine whether LEK1 interacts with the pRb long pocket region in a similar manner to E2Fs, we performed affinity assays using an E2F-binding incompetent GST-Rb fusion protein (GST-Rb-(379�928), amino acid 706 (C->F). Previous studies have shown that this point mutation in the Rb pocket region prevents E2F interaction (18). Our results indicate that, unlike E2F transcription factors, LEK1 is able to recognize and bind to this E2F-binding incompetent GST-Rb construct (Fig. 5C). I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rbl1-1 3 7 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 Fig 5B. Our results demonstrate that LEK1 is not only able to interact with the pRb pocket region, but it readily recognizes and associates with the divergent p107/p130-like pocket region as well (Fig. 5B). I have assumed that cenpf used for this interaction is from mouse. The origin of the cenpf protein is deduced from a section in the introduction "The human proteins, Mitosin and CENP-F, associate with the kinetochore complex during mitosis and display an expression pattern that is dependent on cell cycle progression and independent of developmental stage (11, 12). In contrast, the chicken and mouse LEK proteins, CMF1 and LEK1, respectively, display expression patterns that are regulated during development (13, 14)." Also a section of the sequence for each cenpf from the different species is presented in Figure 1. cenpf-rb1-6 1 5 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association Fig 6A. The recovered complexes were analyzed for the presence of pocket proteins by Western blot using pRb, p107, and p130 antibodies. As shown in Fig. 6A, lanes 2, 4, and 6, LEK1 forms complexes with all three members of the Rb family in vivo. LEK1 forms complexes with all three members of the Rb family in vivo. cenpf-rbl2-1 1 5 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association Fig 6A. The recovered complexes were analyzed for the presence of pocket proteins by Western blot using pRb, p107, and p130 antibodies. As shown in Fig. 6A, lanes 2, 4, and 6, LEK1 forms complexes with all three members of the Rb family in vivo. LEK1 forms complexes with all three members of the Rb family in vivo. cenpf-rbl1-2 1 8 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait physical association physical association Fig 6A. The recovered complexes were analyzed for the presence of pocket proteins by Western blot using pRb, p107, and p130 antibodies. As shown in Fig. 6A, lanes 2, 4, and 6, LEK1 forms complexes with all three members of the Rb family in vivo. LEK1 forms complexes with all three members of the Rb family in vivo. cenpf-rb1-7 1 5 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association Fig 6B. When the reverse experiments were performed using antibodies against pocket proteins (anti-Rb, anti-p107, and anti-p130) to immunoprecipitate endogenous complexes, LEK1 protein was detected in a complex with each of the three pocket proteins, confirming that LEK1 associates with all three members of the Rb family (Fig. 6B, lanes 1�4). LEK1 forms complexes with all three members of the Rb family in vivo. cenpf-rbl1-3 1 5 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Fig 6B. When the reverse experiments were performed using antibodies against pocket proteins (anti-Rb, anti-p107, and anti-p130) to immunoprecipitate endogenous complexes, LEK1 protein was detected in a complex with each of the three pocket proteins, confirming that LEK1 associates with all three members of the Rb family (Fig. 6B, lanes 1�4). LEK1 forms complexes with all three members of the Rb family in vivo. cenpf-rbl2-2 1 5 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association Fig 6B. When the reverse experiments were performed using antibodies against pocket proteins (anti-Rb, anti-p107, and anti-p130) to immunoprecipitate endogenous complexes, LEK1 protein was detected in a complex with each of the three pocket proteins, confirming that LEK1 associates with all three members of the Rb family (Fig. 6B, lanes 1�4). LEK1 forms complexes with all three members of the Rb family in vivo.