bhf-ucl yang-2008-4 in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Yang F., West AP., Allendorph GP., Choe S., Bjorkman PJ., Yang F., West AP., Allendorph GP., Choe S., Bjorkman PJ. Cardiac - Interactions involving cardiac related proteins From methods: Human HJV and neogenin cDNAs; ectodomain of HJV (residues 1-401) with a C-terminal 6x-His tag. The longest neogenin ectodomain construct used DNA encoding the hydrophobic signal sequence (residues 1 – 33), the ectodomain (residues 34 to 1103), and a C-terminal 6x-His tag. Shorter constructs FNIII 5-6 and FNIII 1-6 were made the 1-33 signal sequence was kept, as well as the his tag and the ecodomain tail region was 1062-1103. In contrast, constructs 852-961 (FNIII 5) or 952-1103 (FNIII 6), have C-terminal 6x-His tag, and a gp67 hydrophobic signal. peptide. Plus: The shorter versions, sFNIII 5-6, sFNIII 5, and sFNIII 6, consisted of residues 853-1054, 853-952, and 952-1054, respectively. Recombinant proteins were purified from the supernatants of baculovirus-infected High Five (Hi-5) cells using Ni-NTA and gel filtration chromatography. Human BMP-2 was expressed in E. coli as inclusion bodies, purified and refolded Proteomic fragments of Neo1 regions based on UniProt record sequence with VDLFVI the N-terminal sequence of the peptide after Endoproteinase Glu-C digestion. This used in pull down expt. bjorkman@caltech.edu Biochemistry 2008 Only protein-protein interactions imex curation imex curation yang-2008-5 in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Yang F., West AP., Allendorph GP., Choe S., Bjorkman PJ., Yang F., West AP., Allendorph GP., Choe S., Bjorkman PJ. Cardiac - Interactions involving cardiac related proteins From methods: Human HJV and neogenin cDNAs; ectodomain of HJV (residues 1-401) with a C-terminal 6x-His tag. The longest neogenin ectodomain construct used DNA encoding the hydrophobic signal sequence (residues 1 – 33), the ectodomain (residues 34 to 1103), and a C-terminal 6x-His tag. Shorter constructs FNIII 5-6 and FNIII 1-6 were made the 1-33 signal sequence was kept, as well as the his tag and the ecodomain tail region was 1062-1103. In contrast, constructs 852-961 (FNIII 5) or 952-1103 (FNIII 6), have C-terminal 6x-His tag, and a gp67 hydrophobic signal. peptide. Plus: The shorter versions, sFNIII 5-6, sFNIII 5, and sFNIII 6, consisted of residues 853-1054, 853-952, and 952-1054, respectively. Recombinant proteins were purified from the supernatants of baculovirus-infected High Five (Hi-5) cells using Ni-NTA and gel filtration chromatography. Human BMP-2 was expressed in E. coli as inclusion bodies, purified and refolded Proteomic fragments of Neo1 regions based on UniProt record sequence with VDLFVI the N-terminal sequence of the peptide after Endoproteinase Glu-C digestion. This used in pull down expt. bjorkman@caltech.edu Biochemistry 2008 Only protein-protein interactions imex curation imex curation yang-2008-6 in vitro In vitro sls static light scattering sls predetermined predetermined participant predetermined Yang F., West AP., Allendorph GP., Choe S., Bjorkman PJ., Yang F., West AP., Allendorph GP., Choe S., Bjorkman PJ. Cardiac - Interactions involving cardiac related proteins From methods: Human HJV and neogenin cDNAs; ectodomain of HJV (residues 1-401) with a C-terminal 6x-His tag. The longest neogenin ectodomain construct used DNA encoding the hydrophobic signal sequence (residues 1 – 33), the ectodomain (residues 34 to 1103), and a C-terminal 6x-His tag. Shorter constructs FNIII 5-6 and FNIII 1-6 were made the 1-33 signal sequence was kept, as well as the his tag and the ecodomain tail region was 1062-1103. In contrast, constructs 852-961 (FNIII 5) or 952-1103 (FNIII 6), have C-terminal 6x-His tag, and a gp67 hydrophobic signal. peptide. Plus: The shorter versions, sFNIII 5-6, sFNIII 5, and sFNIII 6, consisted of residues 853-1054, 853-952, and 952-1054, respectively. Recombinant proteins were purified from the supernatants of baculovirus-infected High Five (Hi-5) cells using Ni-NTA and gel filtration chromatography. Human BMP-2 was expressed in E. coli as inclusion bodies, purified and refolded Proteomic fragments of Neo1 regions based on UniProt record sequence with VDLFVI the N-terminal sequence of the peptide after Endoproteinase Glu-C digestion. This used in pull down expt. bjorkman@caltech.edu Biochemistry 2008 Only protein-protein interactions imex curation imex curation neo1_human Neogenin NEO1 IGDCC2 NGN Immunoglobulin superfamily DCC subclass member 2 protein protein human Homo sapiens Human MAAERGARRLLSTPSFWLYCLLLLGRRAPGAAAARSGSAPQSPGASIRTFTPFYFLVEPVDTLSVRGSSVILNCSAYSEPSPKIEWKKDGTFLNLVSDDRRQLLPDGSLFISNVVHSKHNKPDEGYYQCVATVESLGTIISRTAKLIVAGLPRFTSQPEPSSVYAGNNAILNCEVNADLVPFVRWEQNRQPLLLDDRVIKLPSGMLVISNATEGDGGLYRCVVESGGPPKYSDEVELKVLPDPEVISDLVFLKQPSPLVRVIGQDVVLPCVASGLPTPTIKWMKNEEALDTESSERLVLLAGGSLEISDVTEDDAGTYFCIADNGNETIEAQAELTVQAQPEFLKQPTNIYAHESMDIVFECEVTGKPTPTVKWVKNGDMVIPSDYFKIVKEHNLQVLGLVKSDEGFYQCIAENDVGNAQAGAQLIILEHAPATTGPLPSAPRDVVASLVSTRFIKLTWRTPASDPHGDNLTYSVFYTKEGIARERVENTSHPGEMQVTIQNLMPATVYIFRVMAQNKHGSGESSAPLRVETQPEVQLPGPAPNLRAYAASPTSITVTWETPVSGNGEIQNYKLYYMEKGTDKEQDVDVSSHSYTINGLKKYTEYSFRVVAYNKHGPGVSTPDVAVRTLSDVPSAAPQNLSLEVRNSKSIMIHWQPPAPATQNGQITGYKIRYRKASRKSDVTETLVSGTQLSQLIEGLDRGTEYNFRVAALTINGTGPATDWLSAETFESDLDETRVPEVPSSLHVRPLVTSIVVSWTPPENQNIVVRGYAIGYGIGSPHAQTIKVDYKQRYYTIENLDPSSHYVITLKAFNNVGEGIPLYESAVTRPHTDTSEVDLFVINAPYTPVPDPTPMMPPVGVQASILSHDTIRITWADNSLPKHQKITDSRYYTVRWKTNIPANTKYKNANATTLSYLVTGLKPNTLYEFSVMVTKGRRSSTWSMTAHGTTFELVPTSPPKDVTVVSKEGKPKTIIVNWQPPSEANGKITGYIIYYSTDVNAEIHDWVIEPVVGNRLTHQIQELTLDTPYYFKIQARNSKGMGPMSEAVQFRTPKADSSDKMPNDQASGSGGKGSRLPDLGSDYKPPMSGSNSPHGSPTSPLDSNMLLVIIVSVGVITIVVVVIIAVFCTRRTTSHQKKKRAACKSVNGSHKYKGNSKDVKPPDLWIHHERLELKPIDKSPDPNPIMTDTPIPRNSQDITPVDNSMDSNIHQRRNSYRGHESEDSMSTLAGRRGMRPKMMMPFDSQPPQPVISAHPIHSLDNPHHHFHSSSLASPARSHLYHPGSPWPIGTSMSLSDRANSTESVRNTPSTDTMPASSSQTCCTDHQDPEGATSSSYLASSQEEDSGQSLPTAHVRPSHPLKSFAVPAIPPPGPPTYDPALPSTPLLSQQALNHHIHSVKTASIGTLGRSRPPMPVVVPSAPEVQETTRMLEDSESSYEPDELTKEMAHLEGLMKDLNAITTA rgmc_human Hemojuvelin HJV HFE2 Hemochromatosis type 2 protein Hemojuvelin BMP coreceptor RGM domain family member C RGMC protein protein human Homo sapiens Human MGEPGQSPSPRSSHGSPPTLSTLTLLLLLCGHAHSQCKILRCNAEYVSSTLSLRGGGSSGALRGGGGGGRGGGVGSGGLCRALRSYALCTRRTARTCRGDLAFHSAVHGIEDLMIQHNCSRQGPTAPPPPRGPALPGAGSGLPAPDPCDYEGRFSRLHGRPPGFLHCASFGDPHVRSFHHHFHTCRVQGAWPLLDNDFLFVQATSSPMALGANATATRKLTIIFKNMQECIDQKVYQAEVDNLPVAFEDGSINGGDRPGGSSLSIQTANPGNHVEIQAAYIGTTIIIRQTAGQLSFSIKVAEDVAMAFSAEQDLQLCVGGCPPSQRLSRSERNRRGAITIDTARRLCKEGLPVEDAYFHSCVFDVLISGDPNFTVAAQAALEDARAFLPDLEKLHLFPSDAGVPLSSATLLAPLLSGLFVLWLCIQ bmp2_human Bone morphogenetic protein 2 BMP2 BMP2A Bone morphogenetic protein 2A protein protein human Homo sapiens Human MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKQRPTPSRDAVVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEESLEELPETSGKTTRRFFFNLSSIPTEEFITSAELQVFREQMQDALGNNSSFHHRINIYEIIKPATANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEEKQGVSKRHVRISRSLHQDEHSWSQIRPLLVTFGHDGKGHPLHKREKRQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR hjv-neo1-1 1853721 N/A 1853724 unspecified role unspecified role prey prey FNIII-6 fibronectin type III-like 6 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. 1853721 Stoichiometry: 1.0 N/A 1853725 unspecified role unspecified role bait bait purified purified c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Q6ZVN8:p.Asp172Ala asp172ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength 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 trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. 1853721 Stoichiometry: 1.0 direct interaction direct interaction false false false 1853721 Table 1;Figure 2A, B hfe2-neo1-1 1853721 N/A 1853726 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 N/A 1853724 unspecified role unspecified role ancillary ancillary FNIII 5-6 fibronectin type III-like 5-6 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 N/A 1853725 unspecified role unspecified role prey prey c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. physical association physical association false false false 1853721 Figure 4 bmp2-hfe2-1 1853721 N/A 1853726 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 N/A 1853725 unspecified role unspecified role prey prey c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. direct interaction direct interaction false false false 1853721 Figure 4A hfe2-neo1-2 1853722 N/A 1853724 unspecified role unspecified role prey prey FNIII 5-6 fibronectin type III-like 5-6 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 N/A 1853725 unspecified role unspecified role bait bait c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 1853754 1853750 direct interaction direct interaction false false false Figure S1 hfe2-neo1-3 1853723 N/A 1853724 unspecified role unspecified role neutral component neutral component FNIII 5-6 fibronectin type III-like 5-6 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 N/A 1853725 unspecified role unspecified role neutral component neutral component c-terminal c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 direct interaction direct interaction false false false Figure 3