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/ http://www.ebi.ac.uk/ amsili-2007-1 in vitro In vitro pull down pull down sequence tag sequence tag identification sequence tag Amsili S., Zer H., Hinderlich S., Krause S., Becker-Cohen M., MacArthur DG., North KN., Mitrani-Rosenbaum S. Rare diseases - Interactions investigated in the context of Rare genetic disease A freshly prepared lysate from human primary skeletal muscle culture cells was fractionated by anion-exchange chromatography to produce 26 fractions (Figure 1A). Interaction between GNE and the different fractions was tested using SPR technology. Freshly prepared normal and mutant (M712T) GNE proteins were immobilized on a CM5 chip. Two pools of positive fractions (#11–13 and #14–16) were further analyzed by in vitro binding assay.The pooled fractions (#11–13 and #14–16) were incubated with GNE-bound nickel beads, resolved by a gradient SDS-PAGE and visualized by Gelcode reagent staining (Figure 2). The candidate bands, as well as the corresponding gel-areas or bands of the negative-controls, were cut from the gel for analysis by MS. stella@cc.huji.ac.il PloS one 2007 imex curation Only protein-protein interactions imex curation amsili-2007-2 in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Amsili S., Zer H., Hinderlich S., Krause S., Becker-Cohen M., MacArthur DG., North KN., Mitrani-Rosenbaum S. Rare diseases - Interactions investigated in the context of Rare genetic disease from ref 19 alpha-actinin 1 is human stella@cc.huji.ac.il PloS one 2007 imex curation Only protein-protein interactions imex curation amsili-2007-3 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Amsili S., Zer H., Hinderlich S., Krause S., Becker-Cohen M., MacArthur DG., North KN., Mitrani-Rosenbaum S. Rare diseases - Interactions investigated in the context of Rare genetic disease stella@cc.huji.ac.il PloS one 2007 imex curation Only protein-protein interactions imex curation actn1_human Alpha-actinin-1 ACTN1 Alpha-actinin cytoskeletal isoform Non-muscle alpha-actinin-1 F-actin cross-linking protein protein protein human Homo sapiens Human MDHYDSQQTNDYMQPEEDWDRDLLLDPAWEKQQRKTFTAWCNSHLRKAGTQIENIEEDFRDGLKLMLLLEVISGERLAKPERGKMRVHKISNVNKALDFIASKGVKLVSIGAEEIVDGNVKMTLGMIWTIILRFAIQDISVEETSAKEGLLLWCQRKTAPYKNVNIQNFHISWKDGLGFCALIHRHRPELIDYGKLRKDDPLTNLNTAFDVAEKYLDIPKMLDAEDIVGTARPDEKAIMTYVSSFYHAFSGAQKAETAANRICKVLAVNQENEQLMEDYEKLASDLLEWIRRTIPWLENRVPENTMHAMQQKLEDFRDYRRLHKPPKVQEKCQLEINFNTLQTKLRLSNRPAFMPSEGRMVSDINNAWGCLEQVEKGYEEWLLNEIRRLERLDHLAEKFRQKASIHEAWTDGKEAMLRQKDYETATLSEIKALLKKHEAFESDLAAHQDRVEQIAAIAQELNELDYYDSPSVNARCQKICDQWDNLGALTQKRREALERTEKLLETIDQLYLEYAKRAAPFNNWMEGAMEDLQDTFIVHTIEEIQGLTTAHEQFKATLPDADKERLAILGIHNEVSKIVQTYHVNMAGTNPYTTITPQEINGKWDHVRQLVPRRDQALTEEHARQQHNERLRKQFGAQANVIGPWIQTKMEEIGRISIEMHGTLEDQLSHLRQYEKSIVNYKPKIDQLEGDHQLIQEALIFDNKHTNYTMEHIRVGWEQLLTTIARTINEVENQILTRDAKGISQEQMNEFRASFNHFDRDHSGTLGPEEFKACLISLGYDIGNDPQGEAEFARIMSIVDPNRLGVVTFQAFIDFMSRETADTDTADQVMASFKILAGDKNYITMDELRRELPPDQAEYCIARMAPYTGPDSVPGALDYMSFSTALYGESDL ldhb_human L-lactate dehydrogenase B chain LDHB LDH heart subunit Renal carcinoma antigen NY-REN-46 protein protein human Homo sapiens Human MATLKEKLIAPVAEEEATVPNNKITVVGVGQVGMACAISILGKSLADELALVDVLEDKLKGEMMDLQHGSLFLQTPKIVADKDYSVTANSKIVVVTAGVRQQEGESRLNLVQRNVNVFKFIIPQIVKYSPDCIIIVVSNPVDILTYVTWKLSGLPKHRVIGSGCNLDSARFRYLMAEKLGIHPSSCHGWILGEHGDSSVAVWSGVNVAGVSLQELNPEMGTDNDSENWKEVHKMVVESAYEVIKLKGYTNWAIGLSVADLIESMLKNLSRIHPVSTMVKGMYGIENEVFLSLPCILNARGLTSVINQKLKDDEVAQLKKSADTLWDIQKDLKDL glcne_human Bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase GNE GLCNE UDP-GlcNAc-2-epimerase/ManAc kinase protein protein human Homo sapiens Human MEKNGNNRKLRVCVATCNRADYSKLAPIMFGIKTEPEFFELDVVVLGSHLIDDYGNTYRMIEQDDFDINTRLHTIVRGEDEAAMVESVGLALVKLPDVLNRLKPDIMIVHGDRFDALALATSAALMNIRILHIEGGEVSGTIDDSIRHAITKLAHYHVCCTRSAEQHLISMCEDHDRILLAGCPSYDKLLSAKNKDYMSIIRMWLGDDVKSKDYIVALQHPVTTDIKHSIKMFELTLDALISFNKRTLVLFPNIDAGSKEMVRVMRKKGIEHHPNFRAVKHVPFDQFIQLVAHAGCMIGNSSCGVREVGAFGTPVINLGTRQIGRETGENVLHVRDADTQDKILQALHLQFGKQYPCSKIYGDGNAVPRILKFLKSIDLQEPLQKKFCFPPVKENISQDIDHILETLSALAVDLGGTNLRVAIVSMKGEIVKKYTQFNPKTYEERINLILQMCVEAAAEAVKLNCRILGVGISTGGRVNPREGIVLHSTKLIQEWNSVDLRTPLSDTLHLPVWVDNDGNCAALAERKFGQGKGLENFVTLITGTGIGGGIIHQHELIHGSSFCAAELGHLVVSLDGPDCSCGSHGCIEAYASGMALQREAKKLHDEDLLLVEGMSVPKDEAVGALHLIQAAKLGNAKAQSILRTAGTALGLGVVNILHTMNPSLVILSGVLASHYIHIVKDVIRQQALSSVQDVDVVVSDLVDPALLGAASMVLDYTTRRIY gne-actinin-ldhb-1 3531003 N/A 3531006 unspecified role unspecified role prey prey partially purified partially purified human-skeletal-muscle Homo sapiens skeletal muscle. skeletal_muscle Skeletal muscle N/A 3531007 unspecified role unspecified role prey prey partially purified partially purified human-skeletal-muscle Homo sapiens skeletal muscle. skeletal_muscle Skeletal muscle N/A 3531008 unspecified role unspecified role bait bait purified purified His tag 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 false ecoli Escherichia coli (strain K12) association association false false false Fig.1, Fig.2, Table1 act-gne-1 3531004 N/A 3531006 unspecified role unspecified role prey prey purified purified His tag 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 false N/A 3531008 unspecified role unspecified role bait bait purified purified Q9Y223:p.Met712Thr mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false spofr Spodoptera frugiperda (Fall armyworm) Fall armyworm direct interaction direct interaction false false false 3531004 Table2, Fig.3, Fig.4 gne-actn1-1 3531005 N/A 3531006 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level N/A 3531008 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed 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 false physical association physical association false false false Fig. 5B