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/ The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. J. Biol. Chem. (0021-9258) 2007 imex curation Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu 17-April-2008: Contacted by JYOTI. Human kidney cDNA Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu Accepted 2008-MAR-7 by CDEROW. 17-April-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. J. Biol. Chem. (0021-9258) 2007 imex curation Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu 17-April-2008: Contacted by JYOTI. Human kidney cDNA Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. Origin of synaptotagmin 1, SNAP-25, SNAP-23, and syntaxin-4 supplied by the author jsands@emory.edu Accepted 2008-MAR-7 by CDEROW. 17-April-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. J. Biol. Chem. (0021-9258) 2007 imex curation Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu 17-April-2008: Contacted by JYOTI. Human kidney cDNA Only protein-protein interactions canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. MDCK cells are stably infected with rat Slc14a2 jsands@emory.edu Accepted 2008-MAR-7 by CDEROW. 17-April-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. J. Biol. Chem. (0021-9258) 2007 imex curation Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu 17-April-2008: Contacted by JYOTI. Human kidney cDNA Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu Accepted 2008-MAR-7 by CDEROW. 17-April-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. J. Biol. Chem. (0021-9258) 2007 imex curation Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu 17-April-2008: Contacted by JYOTI. Human kidney cDNA Only protein-protein interactions in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu Accepted 2008-MAR-7 by CDEROW. 17-April-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. J. Biol. Chem. (0021-9258) 2007 imex curation Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu 17-April-2008: Contacted by JYOTI. Human kidney cDNA Only protein-protein interactions canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line confocal microscopy confocal microscopy immunostaining immunostaining Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. MDCK cells are stably infected with rat Slc14a2. SNAPAP was detected by tag fluorescence. jsands@emory.edu Accepted 2008-MAR-7 by CDEROW. 17-April-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein. J. Biol. Chem. (0021-9258) 2007 imex curation Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu 17-April-2008: Contacted by JYOTI. Human kidney cDNA Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) 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 Mistry AC., Mallick R., Frohlich O., Klein JD., Rehm A., Chen G., Sands JM. Synapse - Interactions of proteins with an established role in the presynapse. jsands@emory.edu Accepted 2008-MAR-7 by CDEROW. 17-April-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation syt1_human Synaptotagmin-1 Synaptotagmin I p65 SYT1 SYT SVP65 protein protein human Homo sapiens Human MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPWALIAIAIVAVLLVLTCCFCICKKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKVFLLPDKKKKFETKVHRKTLNPVFNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDIIGEFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVVTVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEEVDAMLAVKK DIP protein P21579 original sequence version: 146 467F7C58E411AFA9 snp25_human Synaptosomal-associated protein 25 Synaptosomal-associated 25 kDa protein Super protein SNAP25 SNAP protein protein human Homo sapiens Human MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG DIP protein P60880 original sequence version: 61 DIP protein P60880 original sequence version: 117 FBED2B082A4CB6A6 snp23_rat Synaptosomal-associated protein 23 Snap23 Vesicle-membrane fusion protein SNAP-23 protein protein rat Rattus norvegicus (Rat) Rat MDDLSPEEIQLRAHQVTDESLESTRRILGLAIESQDAGIKTITMLDEQGEQLNRIEEGMDQINKDMREAEKTLTELNKCCGLCVCPCNRTKNFESGKNYKATWGDGGDSSPSNVVSKQPSRITNGQPQQTTGAASGGYIKRITNDAREDEMEENLTQVGSILGNLKNMALDMGNEIDAQNQQIQKITEKADTNKNRIDIANTRAKKLIDS 0D63E3A6F9FE3BA2 snapn_human SNARE-associated protein Snapin SNAPIN SNAP25BP SNAPAP Synaptosomal-associated protein 25-binding protein BLOC1S7 Biogenesis of lysosome-related organelles complex 1 subunit 7 protein protein human Homo sapiens Human MAGAGSAAVSGAGTPVAGPTGRDLFAEGLLEFLRPAVQQLDSHVHAVRESQVELREQIDNLATELCRINEDQKVALDLDPYVKKLLNARRRVVLVNNILQNAQERLRRLNHSVAKETARRRAMLDSGIYPPGSPGK 3EA402AC53C81FFF e2r9t5_canlf Biogenesis of lysosome-related organelles complex 1 subunit 7 SNAPIN protein protein canlf Canis familiaris (dog) Canis lupus familiaris Dog Canis familiaris MTVTEREREEETQAEGEAGSMHREPDVGLDPGSPGSRPGPKAGAKPLRHPGIPFSGSFAININFTSVRCQNALFSKLQTQLFFFFFKPRSGGPGPRPRPRRRGFPEVPRPRPAGSSPAPPGGALQRNSSSRRPARQFPAEGLLVMAAAGSAAVSGAGTPVAGPAGRDLFAEGLLEFLRPAVQQLDSHVHAVRESQVELREQIDNLATELCRINEDQKVALDLDPYVKKLLNARRRVVLVNNILQNAQERLRRLNHSVAKETARRRAMLDSGLYPPGSPSK E47EDF0A5D2F29E2 snp23_human Synaptosomal-associated protein 23 Vesicle-membrane fusion protein SNAP-23 SNAP23 protein protein human Homo sapiens Human MDNLSSEEIQQRAHQITDESLESTRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTELNKCCGLCVCPCNRTKNFESGKAYKTTWGDGGENSPCNVVSKQPGPVTNGQLQQPTTGAASGGYIKRITNDAREDEMEENLTQVGSILGNLKDMALNIGNEIDAQNPQIKRITDKADTNRDRIDIANARAKKLIDS AC378E9786C3A239 stx4_rat Syntaxin-4 Stx4 Stx4a protein protein rat Rattus norvegicus (Rat) Rat MRDRTHELRQGDNISDDEDEVRVALVVHSGAARLSSPDDEFFQKVQTIRQTMAKLESKVRELEKQQVTILATPLPEESMKQGLQNLREEIKQLGREVRAQLKAIEPQKEEADENYNSVNTRMKKTQHGVLSQQFVELINKCNSMQSEYREKNVERIRRQLKITNAGMVSDEELEQMLDSGQSEVFVSNILKDTQVTRQALNEISARHSEIQQLERSIRELHEIFTFLATEVEMQGEMINRIEKNILSSADYVERGQEHVKIALENQKKARKKKVMIAICVSVTVLILAVIIGITITVG E98547758FE6CEC6 ut2_rat Urea transporter 2 Slc14a2 Ut2 Urea transporter, kidney Solute carrier family 14 member 2 protein protein rat Rattus norvegicus (Rat) Rat MSDHPLKEMSDNNRSPPLPEPLSSRYKLYESELSSPTWPSSSQDTHPALPLLEMPEEKDLRSSDEDSHIVKIEKPNERSKRRESELPRRASAGRGAFSLFQAVSYLTGDMKECKNWLKDKPLVLQFLDWVLRGAAQVMFVNNPLSGLIIFIGLLIQNPWWTIAGALGTVVSTLAALALSQDRSAIASGLHGYNGMLVGLLVAVFSEKLDYYWWLLFPVTFASMACPVISSALSTVFAKWDLPVFTLPFNIALTLYLAATGHYNLFFPTTLVKPASSAPNITWSEIEMPLLLQTIPVGVGQVYGCDNPWTGGVILVALFISSPLICLHAAIGSIVGLLAALTVATPFETIYTGLWSYNCVLSCVAIGGMFYVLTWQTHLLALVCALFCAYTGAALSNMMAVVGVPPGTWAFCLSTLTFLLLTSNNPGIHKLPLSKVTYPEANRIYFLTAKRSDEQKPPNGGGGEQSHGGGQRKAEEGSETVFPRRKSVFHIEWSSIRRRSKVFGKSEHQERQTKEPLPYLYRKPTVELLDLNTMEESSEIKVETNTTRTTWIQSSMIAGGKRVSKALSYITGEMKECGEGLKDKSPVFQFLDWVLRGTSQVMFVNNPLSGILIVLGLFVQNPWWAISGCLGTIMSTLTALILSQDKSAIAAGLHGYNGVLVGLLMAVFSDKGNYYWWLLLPVIVMSMTCPILSSALSTVFSKWDLPVFTLPFNIAVTLYLAATGHYNLFFPTKLLQPAVTTPNITWSDVQVPLLLRAIPVGIGQVYGCDNPWTGGIFLVALFVSSPLICLHAAIGSTIGMLAALSIATPFDSIYFGLCGFNSTLACIAIGGMFYVITWQTHLLAIACALFAAYLGAALANMLSVFGLPPCTWPFCLSALTFLLLTTNNPGIYKLPLSKVTYPEANRIYFLSQEKNRRASMITKYQAYDVS 1D12D9D27764AC26 q62668-1 Urea transporter 2 UT-A1 Slc14a2 Ut2 Solute carrier family 14 member 2 Urea transporter, kidney protein protein rat Rattus norvegicus (Rat) Rat MSDHPLKEMSDNNRSPPLPEPLSSRYKLYESELSSPTWPSSSQDTHPALPLLEMPEEKDLRSSDEDSHIVKIEKPNERSKRRESELPRRASAGRGAFSLFQAVSYLTGDMKECKNWLKDKPLVLQFLDWVLRGAAQVMFVNNPLSGLIIFIGLLIQNPWWTIAGALGTVVSTLAALALSQDRSAIASGLHGYNGMLVGLLVAVFSEKLDYYWWLLFPVTFASMACPVISSALSTVFAKWDLPVFTLPFNIALTLYLAATGHYNLFFPTTLVKPASSAPNITWSEIEMPLLLQTIPVGVGQVYGCDNPWTGGVILVALFISSPLICLHAAIGSIVGLLAALTVATPFETIYTGLWSYNCVLSCVAIGGMFYVLTWQTHLLALVCALFCAYTGAALSNMMAVVGVPPGTWAFCLSTLTFLLLTSNNPGIHKLPLSKVTYPEANRIYFLTAKRSDEQKPPNGGGGEQSHGGGQRKAEEGSETVFPRRKSVFHIEWSSIRRRSKVFGKSEHQERQTKEPLPYLYRKPTVELLDLNTMEESSEIKVETNTTRTTWIQSSMIAGGKRVSKALSYITGEMKECGEGLKDKSPVFQFLDWVLRGTSQVMFVNNPLSGILIVLGLFVQNPWWAISGCLGTIMSTLTALILSQDKSAIAAGLHGYNGVLVGLLMAVFSDKGNYYWWLLLPVIVMSMTCPILSSALSTVFSKWDLPVFTLPFNIAVTLYLAATGHYNLFFPTKLLQPAVTTPNITWSDVQVPLLLRAIPVGIGQVYGCDNPWTGGIFLVALFVSSPLICLHAAIGSTIGMLAALSIATPFDSIYFGLCGFNSTLACIAIGGMFYVITWQTHLLAIACALFAAYLGAALANMLSVFGLPPCTWPFCLSALTFLLLTTNNPGIYKLPLSKVTYPEANRIYFLSQEKNRRASMITKYQAYDVS 1D12D9D27764AC26 stx4_human Syntaxin-4 Renal carcinoma antigen NY-REN-31 STX4 STX4A protein protein human Homo sapiens Human MRDRTHELRQGDDSSDEEDKERVALVVHPGTARLGSPDEEFFHKVRTIRQTIVKLGNKVQELEKQQVTILATPLPEESMKQELQNLRDEIKQLGREIRLQLKAIEPQKEEADENYNSVNTRMRKTQHGVLSQQFVELINKCNSMQSEYREKNVERIRRQLKITNAGMVSDEELEQMLDSGQSEVFVSNILKDTQVTRQALNEISARHSEIQQLERSIRELHDIFTFLATEVEMQGEMINRIEKNILSSADYVERGQEHVKTALENQKKARKKKVLIAICVSITVVLLAVIIGVTVVG 5084FD1C49A86BAA q15849-1 Urea transporter 2 UT-A1 SLC14A2 HUT2 UT2 Solute carrier family 14 member 2 Urea transporter, kidney protein protein human Homo sapiens Human MSDPHSSPLLPEPLSSRYKLYEAEFTSPSWPSTSPDTHPALPLLEMPEEKDLRSSNEDSHIVKIEKLNERSKRKDDGVAHRDSAGQRCICLSKAVGYLTGDMKEYRIWLKDKHLALQFIDWVLRGTAQVMFINNPLSGLIIFIGLLIQNPWWTITGGLGTVVSTLTALALGQDRSAIASGLHGYNGMLVGLLMAVFSEKLDYYWWLLFPVTFTAMSCPVLSSALNSIFSKWDLPVFTLPFNIAVTLYLAATGHYNLFFPTTLVEPVSSVPNITWTEMEMPLLLQAIPVGVGQVYGCDNPWTGGVFLVALFISSPLICLHAAIGSIVGLLAALSVATPFETIYTGLWSYNCVLSCIAIGGMFYALTWQTHLLALICALFCAYMEAAISNIMSVVGVPPGTWAFCLATIIFLLLTTNNPAIFRLPLSKVTYPEANRIYYLTVKSGEEEKAPSGGGGEHPPTAGPKVEEGSEAVLSKHRSVFHIEWSSIRRRSKVFGKGEHQERQNKDPFPYRYRKPTVELLDLDTMEESSEIKVETNISKTSWIRSSMAASGKRVSKALSYITGEMKECGEGLKDKSPVFQFFDWVLRGTSQVMFVNNPLSGILIILGLFIQNPWWAISGCLGTIMSTLTALILSQDKSAIAAGFHGYNGVLVGLLMAVFSDKGDYYWWLLLPVIIMSMSCPILSSALGTIFSKWDLPVFTLPFNITVTLYLAATGHYNLFFPTTLLQPASAMPNITWSEVQVPLLLRAIPVGIGQVYGCDNPWTGGIFLIALFISSPLICLHAAIGSTMGMLAALTIATPFDSIYFGLCGFNSTLACIAIGGMFYVITWQTHLLAIACALFAAYLGAALANMLSVFGLPPCTWPFCLSALTFLLLTTNNPAIYKLPLSKVTYPEANRIYYLSQERNRRASIITKYQAYDVS 23BF379DC0767619 slc14a2-snapap-1 4 18 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey physical association physical association Approximately 108 independent clones were screened, and 12 potential L-UT-interacting candidate clones were identified, including snapin. slc14a2-snapap-2 7 18 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey physical association physical association As shown in Fig. 1B (panel c), mating with various baits of UT-A1 and snapin led to expression of the reporter gene, confirming the interaction between snapin and L-UT in yeast. Specificity of the UT-A1-snapin association was shown by transformations of control bait and prey plasmids. Fig. 1B slc14a2-snapap-3 5 18 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 11 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction We demonstrated in vitro binding of recombinant Myc-L-UT and HA-snapin by co-immunoprecipitation using Myc or HA antibody (Fig. 2A) respectively. Fig. 2A slc14a2-snapap-4 5 18 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 11 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 region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction We demonstrated in vitro binding of recombinant Myc-L-UT and HA-snapin by co-immunoprecipitation using Myc or HA antibody (Fig. 2A) respectively. Fig. 2A slc14a2-snapap-5 1 18 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 region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 11 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 region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction Snapin could be co-immunoprecipitated only with L-UT, whereas in the case of N-UT and C-UT, no snapin was detected in the immunoprecipitated complex (Fig. 2B). Fig. 2B slc14a2-snapap-6 2 16 unspecified role unspecified role prey prey rat-kidney_inner_med Rat kidney inner medulla kidney_inner_medulla Kidney inner medulla 11 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction We found that HA-snapin detects native UT-A1, suggesting an in vivo interaction between snapin and UT-A1 in kidney IMCD (Fig. 2C). Fig. 2C slc14a2-snapap-7 2 16 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 11 unspecified role unspecified role bait bait 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 deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction To further characterize the UT-A1-binding site within snapin, we generated six deletion mutants and one PKA phosphomimetic mutant (S50D) of snapin (Fig. 3A) and tested them for their interaction with UT-A1 by GST affinity chromatography (GST pulldown assay). Fig. 3 slc14a2-snapap-8 2 16 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 11 unspecified role unspecified role bait bait 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 ecobb Escherichia coli (strain B / BL21) physical association physical association A strong interaction between GST-snapin and native UT-A1 was detected Fig. 5 MDCK cell line stably expresses rat SLC14A2 Q62668 infection slc14a2-snapap-9 3 16 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait physical association physical association Interaction between snapin and UT-A1 analyzed by coimmunoprecipitations with indicated antibodies from adenovirus-mediated snapin overexpressing UT-A1-MDCK cell lysate. Fig. 6B O95295 infection slc14a2-snapap-10 3 16 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey ecobb Escherichia coli (strain B / BL21) physical association physical association Interaction between snapin and UT-A1 analyzed by coimmunoprecipitations with indicated antibodies from adenovirus-mediated snapin overexpressing UT-A1-MDCK cell lysate. Fig. 6A O95295 infection slc14a2-snapap-11 6 16 unspecified role unspecified role neutral component neutral component 11 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Co-localization of UT-A1 and snapin in UT-A1-MDCK cells. Fig. 7 O95295 transfection slc14a2-snapap-12 2 14 unspecified role unspecified role prey prey rat-kidney_inner_med Rat kidney inner medulla kidney_inner_medulla Kidney inner medulla 11 unspecified role unspecified role bait bait 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 ecobb Escherichia coli (strain B / BL21) 10 unspecified role unspecified role prey prey rat-kidney_inner_med Rat kidney inner medulla kidney_inner_medulla Kidney inner medulla 15 unspecified role unspecified role prey prey rat-kidney_inner_med Rat kidney inner medulla kidney_inner_medulla Kidney inner medulla association association As shown in Fig. 9, snapin detected UT-A1 and t-SNARE components (Syntaxin-4 and SNAP23) in the inner medulla. Fig. 9 slc14a2-syt1-1 2 16 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 12 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 8 unspecified role unspecified role bait bait 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 ecobb Escherichia coli (strain B / BL21) association association Fig. 5 MDCK cell line stably expresses rat SLC14A2 Q62668 infection slc14a2-snap25-1 2 16 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 12 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 9 unspecified role unspecified role bait bait 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 ecobb Escherichia coli (strain B / BL21) association association Fig. 5 MDCK cell line stably expresses rat SLC14A2 Q62668 infection slc14a2-snap23-1 2 16 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 12 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 13 unspecified role unspecified role bait bait 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 ecobb Escherichia coli (strain B / BL21) association association Fig. 5 MDCK cell line stably expresses rat SLC14A2 Q62668 infection slc14a2-stx4-1 2 16 unspecified role unspecified role prey prey 12 unspecified role unspecified role prey prey canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line 17 unspecified role unspecified role bait bait 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 ecobb Escherichia coli (strain B / BL21) association association Fig. 5 MDCK cell line stably expresses rat SLC14A2 Q62668 infection