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/ Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Cell (0092-8674) 2007 imex curation Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 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 bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp Accepted 2007-NOV-18 by DAVET. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Cell (0092-8674) 2007 imex curation Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 rat-neuron Rattus norvegicus neuronal cell neuron Neuron [Neuronal cell; Neural cell anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp Accepted 2007-NOV-18 by DAVET. 13-Nov-2007: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Cell (0092-8674) 2007 imex curation Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 mouse-brain Mus musculus brain brain Brain [cerebrum] anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp Accepted 2007-NOV-18 by DAVET. 13-Nov-2007: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Cell (0092-8674) 2007 imex curation Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 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. pull down pull down anti tag western anti tag western blot anti tag western Iwasato T., Katoh H., Nishimaru H., Ishikawa Y., Inoue H., Saito YM., Ando R., Iwama M., Takahashi R., Negishi M., Itohara S. EphA4 immunoprecipitated with Fc region-fused ephrinA1. Identity of both EphA4 and N-chimaerin confirmed by anti-tag western blotting. iwasato@brain.riken.jp Accepted 2007-NOV-18 by DAVET. 13-Nov-2007: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation epha4_mouse Ephrin type-A receptor 4 Tyrosine-protein kinase receptor SEK-1 Tyrosine-protein kinase receptor MPK-3 Epha4 Sek Sek1 protein protein mouse Mus musculus Mouse MAGIFYFILFSFLFGICDAVTGSRVYPANEVTLLDSRSVQGELGWIASPLEGGWEEVSIMDEKNTPIRTYQVCNVMEASQNNWLRTDWITREGAQRVYIEIKFTLRDCNSLPGVMGTCKETFNLYYYESDNDKERFIRESQFGKIDTIAADESFTQVDIGDRIMKLNTEIRDVGPLSKKGFYLAFQDVGACIALVSVRVFYKKCPLTVRNLAQFPDTITGADTSSLVEVRGSCVNNSEEKDVPKMYCGADGEWLVPIGNCLCNAGHEEQNGECQACKIGYYKALSTDASCAKCPPHSYSVWEGATSCTCDRGFFRADNDAASMPCTRPPSAPLNLISNVNETSVNLEWSSPQNTGGRQDISYNVVCKKCGAGDPSKCRPCGSGVHYTPQQNGLKTTRVSITDLLAHTNYTFEIWAVNGVSKYNPSPDQSVSVTVTTNQAAPSSIALVQAKEVTRYSVALAWLEPDRPNGVILEYEVKYYEKDQNERSYRIVRTAARNTDIKGLNPLTSYVFHVRARTAAGYGDFSEPLEVTTNTVPSRIIGDGANSTVLLVSVSGSVVLVVILIAAFVISRRRSKYSKAKQEADEEKHLNQGVRTYVDPFTYEDPNQAVREFAKEIDASCIKIEKVIGVGEFGEVCSGRLKVPGKREICVAIKTLKAGYTDKQRRDFLSEASIMGQFDHPNIIHLEGVVTKCKPVMIITEYMENGSLDAFLRKNDGRFTVIQLVGMLRGIGSGMKYLSDMSYVHRDLAARNILVNSNLVCKVSDFGMSRVLEDDPEAAYTTRGGKIPIRWTAPEAIAYRKFTSASDVWSYGIVMWEVMSYGERPYWDMSNQDVIKAIEEGYRLPPPMDCPIALHQLMLDCWQKERSDRPKFGQIVNMLDKLIRNPNSLKRTGSESSRPNTALLDPSSPEFSAVVSVGDWLQAIKMDRYKDNFTAAGYTTLEAVVHMSQDDLARIGITAITHQNKILSSVQAMRTQMQQMHGRMVPV DIP protein Q03137 original sequence version: 161 DIP protein Q03137 original sequence version: 101 89BEB2C7CDB54A55 d3zzk3_rat Receptor protein-tyrosine kinase Epha4 protein protein rat Rattus norvegicus (Rat) Rat MAGIFYFILFSFLFGICDAVTGSRVYPANEVTLLDSRSVQGELGWIASPLEGGWEEVSIMDEKNTPIRTYQVCNVMEASQNNWLRTDWITREGAQRVYIEIKFTLRDCNSLPGVMGTCKETFNLYYYESDNDKERFIRESQFGKIDTIAADESFTQVDIGDRIMKLNTEIRDVGPLSKKGFYLAFQDVGACIALVSVRVFYKKCPLTVRNLAQFPDTITGADTSSLVEVRGSCVNNSEEKDVPKMYCGADGEWLVPIGNCLCNAGHEEQNGECQACKIGYYKALSTDATCAKCPPHSYSVWEGATSCTCDRGFFRADNDAASMPCTRPPSAPLNLISNVNETSVNLEWSSPQNTGGRQDISYNVVCKKCGAGDPSKCRPCGSGVHYTPQQNGLKTTRVSITDLLAHTNYTFEIWAVNGVSKYNPSPDQSVSVTVTTNQAAPSSIALVQAKEVTRYSVALAWLEPDRPNGVILEYEVKYYEKDQNERSYRIVRTAARNTDIKGLNPLTSYVFHVRARTAAGYGDFSEPLEVTTNTVPSRIIGDGANSTVLLVSVSGSVVLVVILIAAFVISRRRSKYSQAKQEADEEKHLNQGVRTYVDPFTYEDPNQAVREFAKEIDASCIKIEKVIGVGEFGEVCSGRLKVPGKREICVAIKTLKAGYTDKQRRDFLSEASIMGQFDHPNIIHLEGVVTKCKPVMIITEYMENGSLDAFLRKNDGRFTVIQLVGMLRGIGSGMKYLSDMSYVHRDLAARNILVNSNLVCKVSDFGMSRVLEDDPEAAYTTRGGKIPIRWTAPEAIAYRKFTSASDVWSYGIVMWEVMSYGERPYWDMSNQDVIKAIEEGYRLPPPMDCPIALHQLMLDCWQKERSDRPKFGQIVNMLDKLIRNPNSLKRTGPESSRPNTALLDPSSPEFSAVVSVGDWLQAIKMDRYKDNFTAAGYTTLEAVVHMSQDDLARIGITAITHQNKILSSVQAMRTQMQQMHGRMVPV 77316F0D36349062 chin_mouse N-chimaerin N-chimerin Alpha-chimerin A-chimaerin Rho GTPase-activating protein 2 Chn1 Arhgap2 protein protein mouse Mus musculus Mouse MALTLFDTDEYRPPVWKSYLYQLQQEAPHPRRVTCTCEVENRPKYYGREYHGMISREETDQLLSVAEGSYLIRESQRQPGTYTLALRFGSQTRNFRLYYDGKHFVGEKRFESIHDLVTDGLITLYIETKAAEYIAKMTINPIYEHIGYTTLNREPAYKQHMAVLKETHDEKEATGQDGVSEKRLTSLVRRATLKENEQIPKYEKVHNFKVHTFRGPHWCEYCANFMWGLIAQGVKCADCGLNVHKQCSKMVPNDCKPDLKHVKKVYSCDLTTLVKAHITKRPMVVDMCIREIESRGLNSEGLYRVSGFSDLIEDVKMAFDRDGEKADISVNMYEDINIITGALKLYFRDLPIPLITYDAYPKFIESAKIMDPDEQLETLHEALRSLPPAHCETLRYLMAHLKRVTLHEKENLMSAENLGIVFGPTLMRSPELDPMAALNDIRYQRLVVELLIKNEDILF EE10CCDA8229C0B0 q91v57-1 N-chimaerin Chn1 Arhgap2 A-chimaerin Alpha-chimerin N-chimerin Rho GTPase-activating protein 2 protein protein mouse Mus musculus Mouse MALTLFDTDEYRPPVWKSYLYQLQQEAPHPRRVTCTCEVENRPKYYGREYHGMISREETDQLLSVAEGSYLIRESQRQPGTYTLALRFGSQTRNFRLYYDGKHFVGEKRFESIHDLVTDGLITLYIETKAAEYIAKMTINPIYEHIGYTTLNREPAYKQHMAVLKETHDEKEATGQDGVSEKRLTSLVRRATLKENEQIPKYEKVHNFKVHTFRGPHWCEYCANFMWGLIAQGVKCADCGLNVHKQCSKMVPNDCKPDLKHVKKVYSCDLTTLVKAHITKRPMVVDMCIREIESRGLNSEGLYRVSGFSDLIEDVKMAFDRDGEKADISVNMYEDINIITGALKLYFRDLPIPLITYDAYPKFIESAKIMDPDEQLETLHEALRSLPPAHCETLRYLMAHLKRVTLHEKENLMSAENLGIVFGPTLMRSPELDPMAALNDIRYQRLVVELLIKNEDILF EE10CCDA8229C0B0 chin_rat N-chimaerin Chn1 Arhgap2 Chn N-chimerin Alpha-chimerin A-chimaerin Rho GTPase-activating protein 2 protein protein rat Rattus norvegicus (Rat) Rat MPSKESWSGRKTNRATVHKSKQEGRQQDLLIAALGMKLGSQKSSVTIWQPLKLFAYSQLTSLVRRATLKENEQIPKYEKVHNFKVHTFRGPHWCEYCANFMWGLIAQGVKCADCGLNVHKQCSKMVPNDCKPDLKHVKKVYSCDLTTLVKAHITKRPMVVDMCIREIESRGLNSEGLYRVSGFSDLIEDVKMAFDRDGEKADISVNMYEDINIITGALKLYFRDLPIPLITYDAYPKFIESAKIVDPDEQLETLHEALRSLPPAHCETLRYLMAHLKRVTLHEKENLMSAENLGIVFGPTLMRSPELDPMAALNDIRYQRLVVELLIKNEDILF DIP protein P30337 original sequence version: 102 D4DB599737D171FB q91v57-2 N-chimaerin Chn1 Arhgap2 A-chimaerin Alpha-chimerin N-chimerin Rho GTPase-activating protein 2 protein protein mouse Mus musculus Mouse MPSKESWSGRKANRATVHKAKPEGRQQGLLIAALGMKLGSQKSSVTIWQPLKLFAYSQLTSLVRRATLKENEQIPKYEKVHNFKVHTFRGPHWCEYCANFMWGLIAQGVKCADCGLNVHKQCSKMVPNDCKPDLKHVKKVYSCDLTTLVKAHITKRPMVVDMCIREIESRGLNSEGLYRVSGFSDLIEDVKMAFDRDGEKADISVNMYEDINIITGALKLYFRDLPIPLITYDAYPKFIESAKIMDPDEQLETLHEALRSLPPAHCETLRYLMAHLKRVTLHEKENLMSAENLGIVFGPTLMRSPELDPMAALNDIRYQRLVVELLIKNEDILF 9EC5DA66D58A36C4 epha4-chn1-1 1 8 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 5 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Both alpha1-chimerin and alpha2-chimerin were precipitated with EphA4WT and kinase-dead EphA4FF (Figure 5A), and alpha2-chimerinmfy was also precipitated with EphA4 (Figure S9). Fig. 5A & Supp. Fig. S9 The mffy mutant, lacking 58 amino acids including 291-293, EIE, also precipitated with EphA4. epha4-chn1-2 1 10 unspecified role unspecified role prey prey n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 5 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Both alpha1-chimerin and alpha2-chimerin were precipitated with EphA4WT and kinase-dead EphA4FF (Figure 5A), and alpha2-chimerinmfy was also precipitated with EphA4 (Figure S9). Fig. 5A epha4-chn1-3 4 7 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range certain certain sequence position certain n-terminal 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) 5 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association EphA4 interacted with the α-chimerin C terminus that is common to both alpha1 and alpha2 isoforms. Fig. 5B epha4-chn1-4 2 6 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Endogenous alpha-chimerin and EphA4 interacted in neurons Fig. 5C epha4-chn1-6 3 5 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Finally, we prepared lysates from the developing motor cortex of WT mice and immunoprecipitated them with anti-alpha2-chimerin antibody. EphA4 was again precipitated with alpha2-chimerin (Figure 5D). Fig. 5D The mffy mutant, lacking 58 amino acids including 291-293, EIE, also precipitated with EphA4.