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/ A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. Cancer Cell (1535-6108) 2010 imex curation Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net 21-Dec-2010: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net Accepted 2010-Not a month!-2 by JYOTI. 21-Dec-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2010 jyoti Only protein-protein interactions imex curation A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. Cancer Cell (1535-6108) 2010 imex curation Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net 21-Dec-2010: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net Accepted 2010-Not a month!-2 by JYOTI. 21-Dec-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2010 jyoti Only protein-protein interactions imex curation A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. Cancer Cell (1535-6108) 2010 imex curation Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net 21-Dec-2010: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro emsa supershift electrophoretic mobility supershift assay emsa supershift tag visualisation tag visualisation Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net Accepted 2010-Not a month!-2 by JYOTI. 21-Dec-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2010 jyoti Only protein-protein interactions imex curation A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. Cancer Cell (1535-6108) 2010 imex curation Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net 21-Dec-2010: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa tag visualisation tag visualisation Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net Accepted 2010-Not a month!-2 by JYOTI. 21-Dec-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2010 jyoti Only protein-protein interactions imex curation A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells. Cancer Cell (1535-6108) 2010 imex curation Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net 21-Dec-2010: Contacted by JYOTI. jyoti Only protein-protein interactions human-hct-15 Human colorectal adenocarcinoma cell line HCT-15 hct-15 Human colorectal adenocarcinoma cell line HCT-15 http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CCL-225&Template=cellBiology confocal microscopy confocal microscopy anti tag immunost anti tag immunostaining anti tag immunost Melo SA., Moutinho C., Ropero S., Calin GA., Rossi S., Spizzo R., Fernandez AF., Davalos V., Villanueva A., Montoya G., Yamamoto H., Schwartz S., Esteller M. Cancer - Interactions investigated in the context of cancer The species of RAN used is believed to be human, but this has not been confirmed by the authors. The supplement states EMSA samples were analysed by autoradiography, however this would seem unlikely. On asking, authors omit to mention species of RAN- stating it was made by Sigma. also stated -"according to manufactures as BCL3 strain." I can only find one type of RAN made by Sigma - Human - but NO mention of that strain on the Sigma web site. The authors state that the Streptavidin/Biotin interaction and anti-biotin antibodies were used to detect participants. Pre-let-7a has been confirmed as being human by the authors. mesteller@iconcologia.net Accepted 2010-Not a month!-2 by JYOTI. 21-Dec-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2010 jyoti Only protein-protein interactions imex curation pre-let-7a-1 Human MiRNA-pre-hsa-let-7a-1 ds rna ds rna double stranded ribonucleic acid human Homo sapiens Human UGGGAUGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUUCCUA 577F8A4C0C754DAA xpo5_human Exportin-5 Ran-binding protein 21 XPO5 KIAA1291 RANBP21 protein protein human Homo sapiens Human MAMDQVNALCEQLVKAVTVMMDPNSTQRYRLEALKFCEEFKEKCPICVPCGLRLAEKTQVAIVRHFGLQILEHVVKFRWNGMSRLEKVYLKNSVMELIANGTLNILEEENHIKDALSRIVVEMIKREWPQHWPDMLIELDTLSKQGETQTELVMFILLRLAEDVVTFQTLPPQRRRDIQQTLTQNMERIFSFLLNTLQENVNKYQQVKTDTSQESKAQANCRVGVAALNTLAGYIDWVSMSHITAENCKLLEILCLLLNEQELQLGAAECLLIAVSRKGKLEDRKPLMVLFGDVAMHYILSAAQTADGGGLVEKHYVFLKRLCQVLCALGNQLCALLGADSDVETPSNFGKYLESFLAFTTHPSQFLRSSTQMTWGALFRHEILSRDPLLLAIIPKYLRASMTNLVKMGFPSKTDSPSCEYSRFDFDSDEDFNAFFNSSRAQQGEVMRLACRLDPKTSFQMAGEWLKYQLSTFLDAGSVNSCSAVGTGEGSLCSVFSPSFVQWEAMTLFLESVITQMFRTLNREEIPVNDGIELLQMVLNFDTKDPLILSCVLTNVSALFPFVTYRPEFLPQVFSKLFSSVTFETVEESKAPRTRAVRNVRRHACSSIIKMCRDYPQLVLPNFDMLYNHVKQLLSNELLLTQMEKCALMEALVLISNQFKNYERQKVFLEELMAPVASIWLSQDMHRVLSDVDAFIAYVGTDQKSCDPGLEDPCGLNRARMSFCVYSILGVVKRTCWPTDLEEAKAGGFVVGYTSSGNPIFRNPCTEQILKLLDNLLALIRTHNTLYAPEMLAKMAEPFTKALDMLDAEKSAILGLPQPLLELNDSPVFKTVLERMQRFFSTLYENCFHILGKAGPSMQQDFYTVEDLATQLLSSAFVNLNNIPDYRLRPMLRVFVKPLVLFCPPEHYEALVSPILGPLFTYLHMRLSQKWQVINQRSLLCGEDEAADENPESQEMLEEQLVRMLTREVMDLITVCCVSKKGADHSSAPPADGDDEEMMATEVTPSAMAELTDLGKCLMKHEDVCTALLITAFNSLAWKDTLSCQRTTSQLCWPLLKQVLSGTLLADAVTWLFTSVLKGLQMHGQHDGCMASLVHLAFQIYEALRPRYLEIRAVMEQIPEIQKDSLDQFDCKLLNPSLQKVADKRRKDQFKRLIAGCIGKPLGEQFRKEVHIKNLPSLFKKTKPMLETEVLDNDGGGLATIFEP DIP protein Q9HAV4 original sequence version: 88 3295A17DF7C37602 ran_human GTP-binding nuclear protein Ran GTPase Ran Ras-related nuclear protein Ras-like protein TC4 Androgen receptor-associated protein 24 RAN ARA24 OK/SW-cl.81 protein protein human Homo sapiens Human MAAQGEPQVQFKLVLVGDGGTGKTTFVKRHLTGEFEKKYVATLGVEVHPLVFHTNRGPIKFNVWDTAGQEKFGGLRDGYYIQAQCAIIMFDVTSRVTYKNVPNWHRDLVRVCENIPIVLCGNKVDIKDRKVKAKSIVFHRKKNLQYYDISAKSNYNFEKPFLWLARKLIGDPNLEFVAMPALAPPEVVMDPALAAQYEHDLEVAQTTALPDEDDDL DIP protein P62826 original sequence version: 138 DIP protein P62826 original sequence version: 67 DIP protein P62826 original sequence version: 93 DIP protein P62826 original sequence version: 125 DIP protein P62826 original sequence version: 96 D5C9B7275C34BCE0 gst_ran_xpo5 2 7 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role bait bait n terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) physical association physical association Fig 4A. Both wild-type and mutant forms of XPO5 were able to bind to Ran in a GTP-dependent manner. The addition of pre-let-7a increased the affinity of XPO5WT to bind GST-Ran. bio-pre-let7a_xpo5 5 7 unspecified role unspecified role neutral component neutral component n terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component 3-prime region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Fig 4B. Biotin labeled pre-let-7a was detected in the cytoplasm 1 hr after transfection and disappeared to the cytoplasm 2 hr after co-transfection with WT XP05. there is no way i can add that the miRna molecule was chemically synthesised. let-7a-xp05 4 8 unspecified role unspecified role ancillary ancillary 6 unspecified role unspecified role neutral component neutral component 3 prime region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association Fig 4D, S4B. The biotin labeled pre-let-7a did not bind XPO5Mut in the presence of Ran and GTP. However pre-let-7a formed a readily detectable complex when incubated with XPO5WT. xpo5-let7a 3 7 unspecified role unspecified role neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component 3 prime region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role ancillary ancillary physical association physical association Fig 4D, S4B. The biotin labeled pre-let-7a did not bind XPO5Mut in the presence of Ran and GTP. However pre-let-7a formed a readily detectable complex when incubated with XPO5WT and a supershift could be observed when the antihistidine antibody was added to the reaction. xpo5-let7a-1 1 6 unspecified role unspecified role neutral component neutral component 3 prime region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role ancillary ancillary physical association physical association Fig 4E, S4C. An exothermic reaction takes place between XPO5WT protein and pre-let-7a in the presence of RanGTP, while no reaction takes place between XPO5Mut and pre-let-7a.