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/ Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. J. Biol. Chem. (0021-9258) 2007 imex curation Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA jyoti 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 Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca Accepted 2008-NOV-7 by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 jyoti Only protein-protein interactions imex curation Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. J. Biol. Chem. (0021-9258) 2007 imex curation Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA jyoti Only protein-protein interactions 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 Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca Accepted 2008-NOV-7 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 jyoti Only protein-protein interactions imex curation Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. J. Biol. Chem. (0021-9258) 2007 imex curation Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA jyoti Only protein-protein interactions 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. pull down pull down anti tag western anti tag western blot anti tag western Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. Co2+ chelate resin used to pull down bait proteins. joe.casey@ualberta.ca Accepted 2008-NOV-7 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 jyoti Only protein-protein interactions imex curation Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. J. Biol. Chem. (0021-9258) 2007 imex curation Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA jyoti 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 Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca Accepted 2008-NOV-7 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 jyoti Only protein-protein interactions imex curation Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. J. Biol. Chem. (0021-9258) 2007 imex curation Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA jyoti Only protein-protein interactions 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 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. His-tagged ILK and kAE1 were co-expressed in HEK 293 cells, and radiolabeled with L-[35S]methionine for 1 h, followed by a 24-h chase period. His-tagged ILK and any associated proteins were purified by nickel-affinity resin. Eluted associated proteins were then incubated with anti-Ct AE1 to immunoprecipitate kAE1 and with anti-ILK to immunoprecipitate ILK. Autoradiographs revealed the interaction of ILK and kAE1. joe.casey@ualberta.ca Accepted 2008-NOV-7 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 jyoti Only protein-protein interactions imex curation Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. J. Biol. Chem. (0021-9258) 2007 imex curation Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA jyoti 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 Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca Accepted 2008-NOV-7 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 jyoti Only protein-protein interactions imex curation Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1. J. Biol. Chem. (0021-9258) 2007 imex curation Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. joe.casey@ualberta.ca 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA jyoti Only protein-protein interactions human-kidney Human kidney kidney kidney [Renal] confocal microscopy confocal microscopy immunostaining immunostaining Keskanokwong T., Shandro HJ., Johnson DE., Kittanakom S., Vilas GL., Thorner P., Reithmeier RA., Akkarapatumwong V., Yenchitsomanus PT., Casey JR. I have not annotated the sucrose density gradient data since both proeins only show up in the insoluble fraction i.e. in with all the crud. joe.casey@ualberta.ca Accepted 2008-NOV-7 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Human kidney cDNA J. Biol. Chem. (0021-9258) 2007 jyoti Only protein-protein interactions imex curation parva_human Alpha-parvin Calponin-like integrin-linked kinase-binding protein CH-ILKBP Matrix-remodeling-associated protein 2 Actopaxin PARVA MXRA2 protein protein human Homo sapiens Human MATSPQKSPSVPKSPTPKSPPSRKKDDSFLGKLGGTLARRKKAKEVSELQEEGMNAINLPLSPIPFELDPEDTMLEENEVRTMVDPNSRSDPKLQELMKVLIDWINDVLVGERIIVKDLAEDLYDGQVLQKLFEKLESEKLNVAEVTQSEIAQKQKLQTVLEKINETLKLPPRSIKWNVDSVHAKSLVAILHLLVALSQYFRAPIRLPDHVSIQVVVVQKREGILQSRQIQEEITGNTEALSGRHERDAFDTLFDHAPDKLNVVKKTLITFVNKHLNKLNLEVTELETQFADGVYLVLLMGLLEGYFVPLHSFFLTPDSFEQKVLNVSFAFELMQDGGLEKPKPRPEDIVNCDLKSTLRVLYNLFTKYRNVE DIP protein Q9NVD7 original sequence version: null F48BB5B1E83F8CEF e2r5h4_canlf 59 kDa serine/threonine-protein kinase ILK Beta-integrin-linked kinase ILK-1 ILK-2 Integrin-linked protein kinase p59ILK protein protein canlf Canis familiaris (dog) Canis lupus familiaris Dog Canis familiaris MDDIFTQCREGNAVAVRLWLDNTENDLNQGDDHGFSPLHWACREGRSAVVEMLIMRGARINVMNRGDDTPLHLAASHGHRDIVQKLLQYKADINAVNEHGNVPLHYACFWGQDQVAEDLVANGALVSICNKYGEMPVDKAKAPLRELLRERAEKMGQNLNRIPYKDTFWKGTTRTRPRNGTLNKHSGIDFKQLNFLAKLNENHSGELWKGRWQGNDIVVKVLKVRDWSTRKSRDFNEECPRLRIFSHPNVLPVLGACQSPPAPHPTLITHWMPYGSLYNVLHEGTNFVVDQSQAVKFALDMARGMAFLHTLEPLIPRHALNSRSVMIDEDMTARISMADVKFSFQCPGRMYAPAWVAPEALQKKPEDTNRRSADMWSFAVLLWELVTREVPFADLSNMEIGMKVALEGLRPTIPPGISPHVCKLMKICMNEDPAKRPKFDMIVPILEKMQDK Similar to Q13418 E2CDC2A12211A1C7 nadap_human Kanadaptin SLC4A1AP Kidney anion exchanger adapter protein Solute carrier family 4 anion exchanger member 1 adapter protein Human lung cancer oncogene 3 protein HLC3 protein protein human Homo sapiens Human MLAPLRNAPGREGATSPSPPTDATGSLGEWDVDRNVKTEGWVSKERISKLHRLRMADILSQSETLASQDLSGDFKKPALPVSPAARSKAPASSSSNPEEVQKEGPTALQDSNSGEPDIPPPQPDCGDFRSLQEEQSRPPTAVSSPGGPARAPPYQEPPWGGPATAPYSLETLKGGTILGTRSLKGTSYCLFGRLSGCDVCLEHPSVSRYHAVLQHRASGPDGECDSNGPGFYLYDLGSTHGTFLNKTRIPPRTYCRVHVGHVVRFGGSTRLFILQGPEEDREAESELTVTQLKELRKQQQILLEKKMLGEDSDEEEEMDTSERKINAGSQDDEMGCTWGMGEDAVEDDAEENPIVLEFQQEREAFYIKDPKKALQGFFDREGEELEYEFDEQGHSTWLCRVRLPVDDSTGKQLVAEAIHSGKKKEAMIQCSLEACRILDTLGLLRQEAVSRKRKAKNWEDEDFYDSDDDTFLDRTGLIEKKRLNRMKKAGKIDEKPETFESLVAKLNDAERELSEISERLKASSQVLSESPSQDSLDAFMSEMKSGSTLDGVSRKKLHLRTFELRKEQQRLKGLIKIVKPAEIPELKKTETQTTGAENKAKKLTLPLFGAMKGGSKFKLKTGTVGKLPPKRPELPPTLMRMKDEPEVEEEEEEEEEEEKEKEEHEKKKLEDGSLSRPQPEIEPEAAVQEMRPPTDLTHFKETQTHENMSQLSEEEQNKDYQDCSKTTSLCAGPSASKNEYEKSRGELKKKKTPGPGKLPPTLSSKYPEDDPDYCVWVPPEGQSGDGRTHLNDKYGY EE0F560648DA39EF paxi_human Paxillin PXN protein protein human Homo sapiens Human MDDLDALLADLESTTSHISKRPVFLSEETPYSYPTGNHTYQEIAVPPPVPPPPSSEALNGTILDPLDQWQPSSSRFIHQQPQSSSPVYGSSAKTSSVSNPQDSVGSPCSRVGEEEHVYSFPNKQKSAEPSPTVMSTSLGSNLSELDRLLLELNAVQHNPPGFPADEANSSPPLPGALSPLYGVPETNSPLGGKAGPLTKEKPKRNGGRGLEDVRPSVESLLDELESSVPSPVPAITVNQGEMSSPQRVTSTQQQTRISASSATRELDELMASLSDFKIQGLEQRADGERCWAAGWPRDGGRSSPGGQDEGGFMAQGKTGSSSPPGGPPKPGSQLDSMLGSLQSDLNKLGVATVAKGVCGACKKPIAGQVVTAMGKTWHPEHFVCTHCQEEIGSRNFFERDGQPYCEKDYHNLFSPRCYYCNGPILDKVVTALDRTWHPEHFFCAQCGAFFGPEGFHEKDGKAYCRKDYFDMFAPKCGGCARAILENYISALNTLWHPECFVCRECFTPFVNGSFFEHDGQPYCEVHYHERRGSLCSGCQKPITGRCITAMAKKFHPEHFVCAFCLKQLNKGTFKEQNDKPYCQNCFLKLFC DIP protein P49023 original sequence version: 115 DIP protein P49023 original sequence version: 126 DIP protein P49023 original sequence version: 169 DIP protein P49023 original sequence version: 140 DIP protein P49023 original sequence version: 175 ABF6C0BE5939623F ilk_human Integrin-linked protein kinase ILK-1 ILK-2 59 kDa serine/threonine-protein kinase p59ILK ILK ILK1 ILK2 Beta-integrin-linked kinase protein protein human Homo sapiens Human MDDIFTQCREGNAVAVRLWLDNTENDLNQGDDHGFSPLHWACREGRSAVVEMLIMRGARINVMNRGDDTPLHLAASHGHRDIVQKLLQYKADINAVNEHGNVPLHYACFWGQDQVAEDLVANGALVSICNKYGEMPVDKAKAPLRELLRERAEKMGQNLNRIPYKDTFWKGTTRTRPRNGTLNKHSGIDFKQLNFLTKLNENHSGELWKGRWQGNDIVVKVLKVRDWSTRKSRDFNEECPRLRIFSHPNVLPVLGACQSPPAPHPTLITHWMPYGSLYNVLHEGTNFVVDQSQAVKFALDMARGMAFLHTLEPLIPRHALNSRSVMIDEDMTARISMADVKFSFQCPGRMYAPAWVAPEALQKKPEDTNRRSADMWSFAVLLWELVTREVPFADLSNMEIGMKVALEGLRPTIPPGISPHVCKLMKICMNEDPAKRPKFDMIVPILEKMQDK DIP protein Q13418 original sequence version: 114 DIP protein Q13418 original sequence version: 163 E37DC2AD5311A1C2 slc4a1ap-ilk-1 1 12 unspecified role unspecified role prey prey 10 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 physical association physical association Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. slc4a1ap-ilk-2 4 12 unspecified role unspecified role prey prey 10 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 physical association physical association Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Table 1 slc4a1ap-ilk-4 3 10 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 12 unspecified role unspecified role prey prey 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 physical association physical association Consistent with formation of an ILK�kAE1 complex, kAE1 was present in the eluant, using anti-HA antibody when co-expressed with ILK as detected with anti-HA antibody Fig. 1 slc4a1ap-ilk-5 2 12 unspecified role unspecified role bait bait 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association {Delta}NtILK bound kAE1 efficiently (Fig. 2B, lane 2), indicating that the C-terminal catalytic domain of ILK is sufficient for kAE1 binding. Figs. 1 and 2 An active kinase domain of ILK1 is not required for SLC4A1AP binding slc4a1ap-ilk-6 2 12 unspecified role unspecified role bait bait 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 10 unspecified role unspecified role prey prey c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ch domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Co-immunoprecipitation was carried out using the lysate from HEK 293 cells co-expressing the kAE1 CH domain, with ILK or alone. Anti-His antibody brought down the CH domain when co-expressed with His-tagged ILK (Fig. 4, lane 2) Fig 1, Fig. 4 slc4a1ap-ilk-7 5 10 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 12 unspecified role unspecified role prey prey 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 region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Autoradiographs revealed the interaction of ILK and kAE1 in HEK 293 cells was detected immediately after the 1-h pulse (Fig. 7B). Fig. 7 slc4a1ap-ilk-8 3 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role prey prey 12 unspecified role unspecified role bait bait 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 11 unspecified role unspecified role prey prey physical association physical association These immunoblots revealed that endogenous paxillin and actopaxin associated with the complex of ILK�kAE1 (Fig. 8, lane 2) Fig. 8 Paxillin or actopaxin were not detected when kAE1 was expressed without ILK suggesting that kAE1 interacts with the actin cytoskeleton through a complex containing ILK, paxillin, and actopaxin. slc4a1ap-ilk-9 6 10 unspecified role unspecified role neutral component neutral component c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 9 unspecified role unspecified role neutral component neutral component colocalization colocalization Co-localization of ILK and kAE1 Fig. 10 Colocalisation only observed in polarised cells slc4a1ap-ilk-10 7 12 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range colocalization colocalization Co-localization of ILK and kAE1 Fig. 11