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/ ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. Biochem. Biophys. Res. Commun. (0006-291X) 2003 imex curation Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer shlu@public.bta.net.cn A human fetal liver yeast two-hybrid cDNA library was used. Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer It was found that ECGR2 is the same as SPINK7 by using SRS to search (All Text) for the GenBank Accession No. AF268198 given for the ECGR2 gene in the introduction. shlu@public.bta.net.cn Accepted 2007-JAN-23 by JYOTI. A human fetal liver yeast two-hybrid cDNA library was used. Biochem. Biophys. Res. Commun. (0006-291X) 2003 Only protein-protein interactions imex curation ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. Biochem. Biophys. Res. Commun. (0006-291X) 2003 imex curation Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer shlu@public.bta.net.cn A human fetal liver yeast two-hybrid cDNA library was used. Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer shlu@public.bta.net.cn Accepted 2007-JAN-23 by JYOTI. A human fetal liver yeast two-hybrid cDNA library was used. Biochem. Biophys. Res. Commun. (0006-291X) 2003 Only protein-protein interactions imex curation ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. Biochem. Biophys. Res. Commun. (0006-291X) 2003 imex curation Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer shlu@public.bta.net.cn A human fetal liver yeast two-hybrid cDNA library was used. Only protein-protein interactions human-ec109 Homo sapiens esophageal cancer cell line ec109 human esophageal cancer cell line confocal microscopy confocal microscopy immunostaining immunostaining Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer shlu@public.bta.net.cn Accepted 2007-JAN-23 by JYOTI. A human fetal liver yeast two-hybrid cDNA library was used. Biochem. Biophys. Res. Commun. (0006-291X) 2003 Only protein-protein interactions imex curation ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. Biochem. Biophys. Res. Commun. (0006-291X) 2003 imex curation Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer shlu@public.bta.net.cn A human fetal liver yeast two-hybrid cDNA library was used. Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Cui Y., Wang J., Zhang X., Lang R., Bi M., Guo L., Lu SH. Cancer - Interactions investigated in the context of cancer shlu@public.bta.net.cn Accepted 2007-JAN-23 by JYOTI. A human fetal liver yeast two-hybrid cDNA library was used. Biochem. Biophys. Res. Commun. (0006-291X) 2003 Only protein-protein interactions imex curation mt1g_human Metallothionein-1G Metallothionein-IG Metallothionein-1K MT1G MT1K MT1M protein protein human Homo sapiens Human MDPNCSCAAAGVSCTCASSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASEKCSCCA A1B390F5899DD040 q8izw4_human protein protein human Homo sapiens Human RRARCWHKAPERQRQGLKPGFSWAGADAQSGVGSCHLGNCWDLQSQRCHLHSMVLILPASWPEGHPDHKSGGLSVCLHLPQRCLLSFPTHSSLFGVRGGTGQLAQDLLIIAAQVFE B48F55470F01DBB0 mt1h_human Metallothionein-1H MT1H Metallothionein-IH Metallothionein-0 protein protein human Homo sapiens Human MDPNCSCEAGGSCACAGSCKCKKCKCTSCKKSCCSCCPLGCAKCAQGCICKGASEKCSCCA 03D866E4F437A2CC isk7_human Serine protease inhibitor Kazal-type 7 Esophagus cancer-related gene 2 protein SPINK7 ECG2 UNQ745/PRO1474 protein protein human Homo sapiens Human MKITGGLLLLCTVVYFCSSSEAASLSPKKVDCSIYKKYPVVAIPCPITYLPVCGSDYITYGNECHLCTESLKSNGRVQFLHDGSC AE861AC8E1EA07D3 rt12_human 28S ribosomal protein S12, mitochondrial MT-RPS12 MRPS12 RPMS12 RPSM12 Mitochondrial small ribosomal subunit protein uS12m protein protein human Homo sapiens Human MSWSGLLHGLNTSLTCGPALVPRLWATCSMATLNQMHRLGPPKRPPRKLGPTEGRPQLKGVVLCTFTRKPKKPNSANRKCCRVRLSTGREAVCFIPGEGHTLQEHQIVLVEGGRTQDLPGVKLTVVRGKYDCGHVQKK 893FD2CA1C7187A7 mt2_human Metallothionein-2 Metallothionein-II Metallothionein-2A MT2A CES1 MT2 protein protein human Homo sapiens Human MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA 7B0A7E1F99843078 epb42_human Protein 4.2 EPB42 E42P Erythrocyte membrane protein band 4.2 protein protein human Homo sapiens Human MGQALGIKSCDFQAARNNEEHHTKALSSRRLFVRRGQPFTIILYFRAPVRAFLPALKKVALTAQTGEQPSKINRTQATFPISSLGDRKWWSAVVEERDAQSWTISVTTPADAVIGHYSLLLQVSGRKQLLLGQFTLLFNPWNREDAVFLKNEAQRMEYLLNQNGLIYLGTADCIQAESWDFGQFEGDVIDLSLRLLSKDKQVEKWSQPVHVARVLGALLHFLKEQRVLPTPQTQATQEGALLNKRRGSVPILRQWLTGRGRPVYDGQAWVLAAVACTVLRCLGIPARVVTTFASAQGTGGRLLIDEYYNEEGLQNGEGQRGRIWIFQTSTECWMTRPALPQGYDGWQILHPSAPNGGGVLGSCDLVPVRAVKEGTLGLTPAVSDLFAAINASCVVWKCCEDGTLELTDSNTKYVGNNISTKGVGSDRCEDITQNYKYPEGSLQEKEVLERVEKEKMEREKDNGIRPPSLETASPLYLLLKAPSSLPLRGDAQISVTLVNHSEQEKAVQLAIGVQAVHYNGVLAAKLWRKKLHLTLSANLEKIITIGLFFSNFERNPPENTFLRLTAMATHSESNLSCFAQEDIAICRPHLAIKMPEKAEQYQPLTASVSLQNSLDAPMEDCVISILGRGLIHRERSYRFRSVWPENTMCAKFQFTPTHVGLQRLTVEVDCNMFQNLTNYKSVTVVAPELSA 38225C311E478580 rabl6_human Rab-like protein 6 RABL6 C9orf86 GTP-binding protein Parf PARF Partner of ARF Rab-like protein 1 protein protein human Homo sapiens Human MFSALKKLVGSDQAPGRDKNIPAGLQSMNQALQRRFAKGVQYNMKIVIRGDRNTGKTALWHRLQGRPFVEEYIPTQEIQVTSIHWSYKTTDDIVKVEVWDVVDKGKCKKRGDGLKMENDPQEAESEMALDAEFLDVYKNCNGVVMMFDITKQWTFNYILRELPKVPTHVPVCVLGNYRDMGEHRVILPDDVRDFIDNLDRPPGSSYFRYAESSMKNSFGLKYLHKFFNIPFLQLQRETLLRQLETNQLDMDATLEELSVQQETEDQNYGIFLEMMEARSRGHASPLAANGQSPSPGSQSPVVPAGAVSTGSSSPGTPQPAPQLPLNAAPPSSVPPVPPSEALPPPACPSAPAPRRSIISRLFGTSPATEAAPPPPEPVPAAEGPATVQSVEDFVPDDRLDRSFLEDTTPARDEKKVGAKAAQQDSDSDGEALGGNPMVAGFQDDVDLEDQPRGSPPLPAGPVPSQDITLSSEEEAEVAAPTKGPAPAPQQCSEPETKWSSIPASKPRRGTAPTRTAAPPWPGGVSVRTGPEKRSSTRPPAEMEPGKGEQASSSESDPEGPIAAQMLSFVMDDPDFESEGSDTQRRADDFPVRDDPSDVTDEDEGPAEPPPPPKLPLPAFRLKNDSDLFGLGLEEAGPKESSEEGKEGKTPSKEKKKKKKKGKEEEEKAAKKKSKHKKSKDKEEGKEERRRRQQRPPRSRERTAADELEAFLGGGAPGGRHPGGGDYEEL 6C3FDB38C7FD7D59 spink7-mt2a 2 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Table 1 Using the fulllength ECRG2 cDNA as bait to screen a human fetal liver cDNA library, we found that nine positive clones could bind to the ECRG2 protein. Here we report one candidate clone, which encoded human metallothionein2A (MT2A). spink7-q8izw4 2 8 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association Table 1 The genes identified by the yeast two-hybrid approach were metallothionein 2A (MT2A), metallothionein 1H, metallothionein 1G, ferritin, erythrocyte membrane protein band 4.2, hemoglobin, mitochondrial ribosomal protein S12, hypothetical protein FLJ10101, and a novel gene whose cDNA was found to have no strong homology to any other previously characterized protein and Gen- Bank Accession No. is AF422192. spink7-mt1h 2 7 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Table 1 The genes identified by the yeast two-hybrid approach were metallothionein 2A (MT2A), metallothionein 1H, spink7-mt1g 2 8 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association Table 1 The genes identified by the yeast two-hybrid approach were metallothionein 2A (MT2A), metallothionein 1H, metallothionein 1G, spink7-ebp42 2 11 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Table 1 The genes identified by the yeast two-hybrid approach were metallothionein 2A (MT2A), metallothionein 1H, metallothionein 1G, ferritin, erythrocyte membrane protein band 4.2, spink7-mrps12 2 8 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association Table 1 The genes identified by the yeast two-hybrid approach were metallothionein 2A (MT2A), metallothionein 1H, metallothionein 1G, ferritin, erythrocyte membrane protein band 4.2, hemoglobin, mitochondrial ribosomal protein S12, spink7-parf 2 12 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Table 1 The genes identified by the yeast two-hybrid approach were metallothionein 2A (MT2A), metallothionein 1H, metallothionein 1G, ferritin, erythrocyte membrane protein band 4.2, hemoglobin, mitochondrial ribosomal protein S12, hypothetical protein FLJ10101, Bernd: replaced fragment Q9NWD6/FLJ10101 (not integrated in SP) by Q3YEC7 (seq identity). spink7-mt2a-1 1 10 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 in vitro In vitro 8 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction 1 C MT2A was bound to GST-ECRG2 mt2a-spink7 4 10 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 A FLAG-ECRG2 was present in immunoprecipitates from HA-MT2A transfected cells spink7-mt2a-2 4 10 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 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 B This immunoblot again showed that the immunoprecipitate of FLAG-ECRG2 contained HA-MT2A spink7-mt2a-3 3 10 unspecified role unspecified role 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 8 unspecified role unspecified role neutral component neutral component region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization 3 C ECRG2-null EC109 cells were transiently co-transfected with plasmids encoding GFP-tagged ECRG2, HA-tagged MT2A, or both of the two plasmids. Immunofluorescence and confocal laser microscopy showed that these two proteins were all localized mostly to nuclei and slightly to cytoplasm