UniProt UniProt http://www.uniprot.org Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 imex curation Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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 Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu Accepted 2019-MAY-06 AT 14:52 BST AT 14:52 BST by MGT The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 Only protein-protein interactions imex curation imex curation Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 imex curation Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu Accepted 2019-MAY-06 AT 14:52 BST AT 14:52 BST by MGT The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 Only protein-protein interactions imex curation imex curation Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 imex curation Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu Accepted 2019-MAY-06 AT 14:52 BST AT 14:52 BST by MGT The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 Only protein-protein interactions imex curation imex curation Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion. The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 imex curation Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation mouse-neuron Mus musculus neuronal cell neuron Neuron [Neuronal cell; Neural cell confocal microscopy confocal microscopy immunocytochemistry immunocytochemistry Shao Q., Yang T., Huang H., Alarmanazi F., Liu G. Guofa.Liu@utoledo.edu Accepted 2019-MAY-06 AT 14:52 BST AT 14:52 BST by MGT The Journal of neuroscience : the official journal of the Society for Neuroscience 2017 Only protein-protein interactions imex curation imex curation tbb3_mouse Tubulin beta-3 chain Tubb3 protein protein mouse Mus musculus Mouse MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPSGNYVGDSDLQLERISVYYNEASSHKYVPRAILVDLEPGTMDSVRSGAFGHLFRPDNFIFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKECENCDCLQGFQLTHSLGGGTGSGMGTLLISKVREEYPDRIMNTFSVVPSPKVSDTVVEPYNATLSIHQLVENTDETYCIDNEALYDICFRTLKLATPTYGDLNHLVSATMSGVTTSLRFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTARGSQQYRALTVPELTQQMFDAKNMMAACDPRHGRYLTVATVFRGRMSMKEVDEQMLAIQSKNSSYFVEWIPNNVKVAVCDIPPRGLKMSSTFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATAEEEGEMYEDDDEESEAQGPK DIP protein Q9ERD7 original sequence version: 82 4B9D9B7DBA102949 tbb3_human Tubulin beta-3 chain TUBB3 TUBB4 Tubulin beta-III Tubulin beta-4 chain protein protein human Homo sapiens Human MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPSGNYVGDSDLQLERISVYYNEASSHKYVPRAILVDLEPGTMDSVRSGAFGHLFRPDNFIFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKECENCDCLQGFQLTHSLGGGTGSGMGTLLISKVREEYPDRIMNTFSVVPSPKVSDTVVEPYNATLSIHQLVENTDETYCIDNEALYDICFRTLKLATPTYGDLNHLVSATMSGVTTSLRFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTARGSQQYRALTVPELTQQMFDAKNMMAACDPRHGRYLTVATVFRGRMSMKEVDEQMLAIQSKNSSYFVEWIPNNVKVAVCDIPPRGLKMSSTFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATAEEEGEMYEDDEEESEAQGPK DIP protein Q13509 original sequence version: 137 4B9CDE7DBA102949 unc5c_human Netrin receptor UNC5C UNC5C Protein unc-5 homolog 3 Protein unc-5 homolog C UNC5H3 protein protein human Homo sapiens Human MRKGLRATAARCGLGLGYLLQMLVLPALALLSASGTGSAAQDDDFFHELPETFPSDPPEPLPHFLIEPEEAYIVKNKPVNLYCKASPATQIYFKCNSEWVHQKDHIVDERVDETSGLIVREVSIEISRQQVEELFGPEDYWCQCVAWSSAGTTKSRKAYVRIAYLRKTFEQEPLGKEVSLEQEVLLQCRPPEGIPVAEVEWLKNEDIIDPVEDRNFYITIDHNLIIKQARLSDTANYTCVAKNIVAKRKSTTATVIVYVNGGWSTWTEWSVCNSRCGRGYQKRTRTCTNPAPLNGGAFCEGQSVQKIACTTLCPVDGRWTPWSKWSTCGTECTHWRRRECTAPAPKNGGKDCDGLVLQSKNCTDGLCMQTAPDSDDVALYVGIVIAVIVCLAISVVVALFVYRKNHRDFESDIIDSSALNGGFQPVNIKAARQDLLAVPPDLTSAAAMYRGPVYALHDVSDKIPMTNSPILDPLPNLKIKVYNTSGAVTPQDDLSEFTSKLSPQMTQSLLENEALSLKNQSLARQTDPSCTAFGSFNSLGGHLIVPNSGVSLLIPAGAIPQGRVYEMYVTVHRKETMRPPMDDSQTLLTPVVSCGPPGALLTRPVVLTMHHCADPNTEDWKILLKNQAAQGQWEDVVVVGEENFTTPCYIQLDAEACHILTENLSTYALVGHSTTKAAAKRLKLAIFGPLCCSSLEYSIRVYCLDDTQDALKEILHLERQMGGQLLEEPKALHFKGSTHNLRLSIHDIAHSLWKSKLLAKYQEIPFYHVWSGSQRNLHCTFTLERFSLNTVELVCKLCVRQVEGEGQIFQLNCTVSEEPTGIDLPLLDPANTITTVTGPSAFSIPLPIRQKLCSSLDAPQTRGHDWRMLAHKLNLDRYLNYFATKSSPTGVILDLWEAQNFPDGNLSMLAAVLEEMGRHETVVSLAAEGQY DIP protein O95185 original sequence version: 67 98A95995129532A7 unc5c_mouse Netrin receptor UNC5C Protein unc-5 homolog 3 Protein unc-5 homolog C Rostral cerebellar malformation protein Unc5c Rcm Unc5h3 protein protein mouse Mus musculus Mouse MRKGLRATAARCGLGLGYLLQMLVLPALALLSASGTGSAAQDDEFFHELPETFPSDPPEPLPHFLIEPEEAYIVKNKPVNLYCKASPATQIYFKCNSEWVHQKDHVVDERVDETSGLIVREVSIEISRQQVEELFGPEDYWCQCVAWSSAGTTKSRKAYVRIAYLRKTFEQEPLGKEVSLEQEVLLQCRPPEGIPVAEVEWLKNEDIIDPAEDRNFYITIDHNLIIKQARLSDTANYTCVAKNIVAKRKSTTATVIVYVNGGWSTWTEWSVCNSRCGRGYQKRTRTCTNPAPLNGGAFCEGQSVQKIACTTLCPVDGRWTSWSKWSTCGTECTHWRRRECTAPAPKNGGKDCDGLVLQSKNCTDGLCMQAAPDSDDVALYVGIVIAVTVCLAITVVVALFVYRKNHRDFESDIIDSSALNGGFQPVNIKAARQDLLAVPPDLTSAAAMYRGPVYALHDVSDKIPMTNSPILDPLPNLKIKVYNSSGAVTPQDDLAEFSSKLSPQMTQSLLENEALNLKNQSLARQTDPSCTAFGTFNSLGGHLIIPNSGVSLLIPAGAIPQGRVYEMYVTVHRKENMRPPMEDSQTLLTPVVSCGPPGALLTRPVILTLHHCADPSTEDWKIQLKNQAVQGQWEDVVVVGEENFTTPCYIQLDAEACHILTENLSTYALVGQSTTKAAAKRLKLAIFGPLCCSSLEYSIRVYCLDDTQDALKEVLQLERQMGGQLLEEPKALHFKGSIHNLRLSIHDIAHSLWKSKLLAKYQEIPFYHIWSGSQRNLHCTFTLERLSLNTVELVCKLCVRQVEGEGQIFQLNCTVSEEPTGIDLPLLDPASTITTVTGPSAFSIPLPIRQKLCSSLDAPQTRGHDWRMLAHKLNLDRYLNYFATKSSPTGVILDLWEAQNFPDGNLSMLAAVLEEMGRHETVVSLAAEGQY 8A5D951A4EECA179 tubb3-unc5c-1 1 5 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level 8 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 1B Netrin-1 To determine whether endogenous TUBB3 interacted with UNC5C, we performed coimmunoprecipitation on the lysates of dissociated cerebellar neurons from postnatal day 4 (P4) mice unc5c-tubb3-1 1 5 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level 8 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 1B, 1D-F, 3A Netrin-1 Pretreatment with Taxol or nocodazole abolished the ability of netrin-1 to reduce the TUBB3/UNC5C interaction unc5c-tubb3-2 3 6 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 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 7 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig. 1A TUBB3 was readily detected in the lysates immunoprecipitated with anti-HA antibody (UNC5C) (Fig. 1A), unc5c-tubb3-3 2 6 unspecified role unspecified role prey prey purified purified n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait purified purified 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 region sufficient to bind sufficient binding region sufficient to bind range range certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association Fig. 1G To further determine whether UNC5C directly interacts with TUBB3, a truncated protein containing only the intracellular and transmembrane domains of UNC5C (UNC5C-ICD-GST) was purified and incubated with purified TUBB3. tubb3-unc5c-2 4 5 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 8 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level colocalization colocalization Fig. 2 Netrin-1 Quantitative colocalization analysis of confocal fluorescence microscopy images showed partial overlap of immunofluorescent signals of UNC5C and TUBB3 in the peripheral region of GCs, including lamellipodia and filopodia (Fig. 2A–C, quantification in J), suggesting that UNC5C partially colocalized with TUBB3 in the GC.