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/ li-2009-48 yeast Saccharomyces cerevisiae 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 predetermined predetermined participant predetermined Li Wq., Shi L., You Yg., Gong Yh., Yin B., Yuan Jg., Peng Xz. pengxiaozhong@pumc.edu.cn BMC biology 2009 Only protein-protein interactions imex curation imex curation li-2009-49 in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Li Wq., Shi L., You Yg., Gong Yh., Yin B., Yuan Jg., Peng Xz. pengxiaozhong@pumc.edu.cn BMC biology 2009 Only protein-protein interactions imex curation imex curation li-2009-50 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 Li Wq., Shi L., You Yg., Gong Yh., Yin B., Yuan Jg., Peng Xz. pengxiaozhong@pumc.edu.cn BMC biology 2009 Only protein-protein interactions imex curation imex curation li-2009-51 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 Li Wq., Shi L., You Yg., Gong Yh., Yin B., Yuan Jg., Peng Xz. pengxiaozhong@pumc.edu.cn BMC biology 2009 Only protein-protein interactions imex curation imex curation li-2009-52 mouse-brain-cortex Mus musculus brain cortex brain_cortex Brain cortex [Cerebral cortex; Cerebrum cortex; Cortex; Pallium] confocal microscopy confocal microscopy immunocytochemistry immunocytochemistry Li Wq., Shi L., You Yg., Gong Yh., Yin B., Yuan Jg., Peng Xz. pengxiaozhong@pumc.edu.cn BMC biology 2009 Only protein-protein interactions imex curation imex curation li-2009-53 mouse-neuron Mus musculus 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 Li Wq., Shi L., You Yg., Gong Yh., Yin B., Yuan Jg., Peng Xz. cortical neurons pengxiaozhong@pumc.edu.cn BMC biology 2009 Only protein-protein interactions imex curation imex curation ntrk3_human NT-3 growth factor receptor NTRK3 TRKC GP145-TrkC Neurotrophic tyrosine kinase receptor type 3 TrkC tyrosine kinase protein protein human Homo sapiens Human MDVSLCPAKCSFWRIFLLGSVWLDYVGSVLACPANCVCSKTEINCRRPDDGNLFPLLEGQDSGNSNGNASINITDISRNITSIHIENWRSLHTLNAVDMELYTGLQKLTIKNSGLRSIQPRAFAKNPHLRYINLSSNRLTTLSWQLFQTLSLRELQLEQNFFNCSCDIRWMQLWQEQGEAKLNSQNLYCINADGSQLPLFRMNISQCDLPEISVSHVNLTVREGDNAVITCNGSGSPLPDVDWIVTGLQSINTHQTNLNWTNVHAINLTLVNVTSEDNGFTLTCIAENVVGMSNASVALTVYYPPRVVSLEEPELRLEHCIEFVVRGNPPPTLHWLHNGQPLRESKIIHVEYYQEGEISEGCLLFNKPTHYNNGNYTLIAKNPLGTANQTINGHFLKEPFPESTDNFILFDEVSPTPPITVTHKPEEDTFGVSIAVGLAAFACVLLVVLFVMINKYGRRSKFGMKGPVAVISGEEDSASPLHHINHGITTPSSLDAGPDTVVIGMTRIPVIENPQYFRQGHNCHKPDTYVQHIKRRDIVLKRELGEGAFGKVFLAECYNLSPTKDKMLVAVKALKDPTLAARKDFQREAELLTNLQHEHIVKFYGVCGDGDPLIMVFEYMKHGDLNKFLRAHGPDAMILVDGQPRQAKGELGLSQMLHIASQIASGMVYLASQHFVHRDLATRNCLVGANLLVKIGDFGMSRDVYSTDYYRLFNPSGNDFCIWCEVGGHTMLPIRWMPPESIMYRKFTTESDVWSFGVILWEIFTYGKQPWFQLSNTEVIECITQGRVLERPRVCPKEVYDVMLGCWQREPQQRLNIKEIYKILHALGKATPIYLDILG dok6_human Docking protein 6 DOK6 DOK5L Downstream of tyrosine kinase 6 protein protein human Homo sapiens Human MASNFNDIVKQGYVKIRSRKLGIFRRCWLVFKKASSKGPRRLEKFPDEKAAYFRNFHKVTELHNIKNITRLPRETKKHAVAIIFHDETSKTFACESELEAEEWCKHLCMECLGTRLNDISLGEPDLLAAGVQREQNERFNVYLMPTPNLDIYGECTMQITHENIYLWDIHNAKVKLVMWPLSSLRRYGRDSTWFTFESGRMCDTGEGLFTFQTREGEMIYQKVHSATLAIAEQHERLMLEMEQKARLQTSLTEPMTLSKSISLPRSAYWHHITRQNSVGEIYSLQGHGFGSSKMSRAQTFPSYAPEQSEEAQQPLSRSSSYGFSYSSSLIQ ntf3_human Neurotrophin-3 Neurotrophic factor HDNF Nerve growth factor 2 NTF3 protein protein human Homo sapiens Human MSILFYVIFLAYLRGIQGNNMDQRSLPEDSLNSLIIKLIQADILKNKLSKQMVDVKENYQSTLPKAEAPREPERGGPAKSAFQPVIAMDTELLRQQRRYNSPRVLLSDSTPLEPPPLYLMEDYVGSPVVANRTSRRKRYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT ntrk3_mouse NT-3 growth factor receptor Ntrk3 GP145-TrkC Neurotrophic tyrosine kinase receptor type 3 TrkC TrkC tyrosine kinase protein protein mouse Mus musculus Mouse MDVSLCPAKCSFWRIFLLGSVWLDYVGSVLACPANCVCSKTEINCRRPDDGNLFPLLEGQDSGNSNGNASINITDISRNITSIHIENWRGLHTLNAVDMELYTGLQKLTIKNSGLRNIQPRAFAKNPHLRYINLSSNRLTTLSWQLFQTLSLRELRLEQNFFNCSCDIRWMQLWQEQGEARLDSQSLYCISADGSQLPLFRMNISQCDLPEISVSHVNLTVREGDNAVITCNGSGSPLPDVDWIVTGLQSINTHQTNLNWTNVHAINLTLVNVTSEDNGFTLTCIAENVVGMSNASVALTVYYPPRVVSLVEPEVRLEHCIEFVVRGNPTPTLHWLYNGQPLRESKIIHMDYYQEGEVSEGCLLFNKPTHYNNGNYTLIAKNALGTANQTINGHFLKEPFPESTDFFDFESDASPTPPITVTHKPEEDTFGVSIAVGLAAFACVLLVVLFIMINKYGRRSKFGMKGPVAVISGEEDSASPLHHINHGITTPSSLDAGPDTVVIGMTRIPVIENPQYFRQGHNCHKPDTYVQHIKRRDIVLKRELGEGAFGKVFLAECYNLSPTKDKMLVAVKALKDPTLAARKDFQREAELLTNLQHEHIVKFYGVCGDGDPLIMVFEYMKHGDLNKFLRAHGPDAMILVDGQPRQAKGELGLSQMLHIASQIASGMVYLASQHFVHRDLATRNCLVGANLLVKIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSFGVILWEIFTYGKQPWFQLSNTEVIECITQGRVLERPRVCPKEVYDVMLGCWQREPQQRLNIKEIYKILHALGKATPIYLDILG dok6_mouse Docking protein 6 Downstream of tyrosine kinase 6 Dok6 protein protein mouse Mus musculus Mouse MASNFNDIVKQGYVKIRSRKLGIFRRCWLVFKKASSKGPRRLEKFPDEKAAYFRNFHKVTELHNIKNITRLPRETKKHAVAIIFHDETSKTFACESELEAEEWCKHLCMECLGTRLNDISLGEPDLLAAGVQREQNERFNVYLMPTPNLDIYGECTMQITHENIYLWDIHNAKVKLVMWPLSSLRRYGRDSTWFTFESGRMCDTGEGLFTFQTREGEMIYQKVHSATLAIAEQHERLMLEMEQKARLQTSLTEPMTLSKSISLPRSAYWHHITRQNSVGEIYSLQGHGFGSSKMSRAQTFPSYAPEQSEEAQPPLSRSSSYGFSYSSSLIQ dok6-trkc-1 3437545 N/A 3437551 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell engineered engineered cytosolic domain sufficient to bind sufficient binding region sufficient to bind range range range range false Q16288:p.Lys572Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false Q16288:p.Tyr516Phe mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false Q16288:p.Tyr821Phe mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false Q16288:p.Tyr710Glu 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 Q16288:p.Tyr709Asp 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 Q16288:p.Tyr705Ala 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 N/A 3437552 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell engineered engineered region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 1C, Fig. 1D, Fig. 3B dok6-trkc-2 3437546 N/A 3437553 stimulator stimulator unspecified role unspecified role purified purified ecoli Escherichia coli (strain K12) N/A 3437551 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate engineered engineered Q16288:p.Lys572Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false Q16288:p.Tyr516Phe mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false Q16288:p.Tyr821Phe 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 v5 tag v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false 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. N/A 3437552 unspecified role unspecified role bait bait purified purified 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 false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) association association false false false Fig. 2A, Fig. 3C Consistent with results from the yeast two-hybrid assay findings, the precipitation results proved that the Dok6 PTB domain binds to the activated TrkC receptor strongly in vitro, while no remarkable binding was found between Dok6 and TrkA or TrkB with activation or no activation (Figure (Figure2a,2a, panel 1) dok6-ntf3-1 3437547 N/A 3437553 stimulator stimulator unspecified role unspecified role purified purified ecoli Escherichia coli (strain K12) N/A 3437551 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell engineered engineered v5 tag v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 3437552 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell engineered engineered 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 association association false false false Fig. 2B dok6-ntf3-2 3437548 N/A 3437554 unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level N/A 3437555 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association false false false Fig. 4C dok6-ntrk3-1 3437549 N/A 3437554 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell N/A 3437555 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell colocalization colocalization false false false Fig. 4B Immunostaining showed that TrkC and Dok6 colocalise at the membrane, cytoplasm and neurites in primary cultured cortex neurons (Figure 4b) dok6-ntrk3-2 3437550 N/A 3437553 stimulator stimulator unspecified role unspecified role purified purified N/A 3437554 unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level N/A 3437555 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association false false false Fig. 6A Consistent with previous results, Dok6 binds to the TrkC receptor in cortex neurons upon NT-3 stimulation, while without NT-3 stimulation no obvious interactions exist (Figure (Figure6a).6a).