mbinfo MBInfo http://www.mechanobio.info/ http://www.mechanobio.info/ li-1999c-1 Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats. in vitro In vitro elisa enzyme linked immunosorbent assay ELISA ELISA elisa antibody detection identification by antibody antibody detection Li F., Zhang Y., Wu C. The source of His-PINCH specified by the author and the source of GST-ILK traced from references. cwu@cellbio.bhs.uab.edu J. Cell. Sci. (0021-9533) 1999 Only protein-protein interactions imex curation li-1999c-2 Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats. crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Li F., Zhang Y., Wu C. The source of His-PINCH specified by the author and the source of GST-ILK traced from references. cwu@cellbio.bhs.uab.edu J. Cell. Sci. (0021-9533) 1999 Only protein-protein interactions imex curation ilk_mouse Integrin-linked protein kinase 59 kDa serine/threonine-protein kinase Beta-integrin-linked kinase ILK-1 ILK-2 ILK1 ILK2 p59ILK Ilk protein protein mouse Mus musculus Mouse MDDIFTQCREGNAVAVRLWLDNTENDLNQGDDHGFSPLHWACREGRSAVVEMLIMRGARINVMNRGDDTPLHLAASHGHRDIVQKLLQYKADINAVNEHGNVPLHYACFWGQDQVAEDLVANGALVSICNKYGEMPVDKAKAPLRELLRERAEKMGQNLNRIPYKDTFWKGTTRTRPRNGTLNKHSGIDFKQLNFLAKLNENHSGELWKGRWQGNDIVVKVLKVRDWSTRKSRDFNEECPRLRIFSHPNVLPVLGACQAPPAPHPTLITHWMPYGSLYNVLHEGTNFVVDQSQAVKFALDMARGMAFLHTLEPLIPRHALNSRSVMIDEDMTARISMADVKFSFQCPGRMYAPAWVAPEALQKKPEDTNRRSADMWSFAVLLWELVTREVPFADLSNMEIGMKVALEGLRPTIPPGISPHVCKLMKICMNEDPAKRPKFDMIVPILEKMQDK lims1_human LIM and senescent cell antigen-like-containing domain protein 1 LIMS1 Particularly interesting new Cys-His protein 1 Renal carcinoma antigen NY-REN-48 PINCH PINCH1 protein protein human Homo sapiens Human MANALASATCERCKGGFAPAEKIVNSNGELYHEQCFVCAQCFQQFPEGLFYEFEGRKYCEHDFQMLFAPCCHQCGEFIIGRVIKAMNNSWHPECFRCDLCQEVLADIGFVKNAGRHLCRPCHNREKARGLGKYICQKCHAIIDEQPLIFKNDPYHPDHFNCANCGKELTADARELKGELYCLPCHDKMGVPICGACRRPIEGRVVNAMGKQWHVEHFVCAKCEKPFLGHRHYERKGLAYCETHYNQLFGDVCFHCNRVIEGDVVSALNKAWCVNCFACSTCNTKLTLKNKFVEFDMKPVCKKCYEKFPLELKKRLKKLAETLGRK nck2_human Cytoplasmic protein NCK2 NCK adaptor protein 2 SH2/SH3 adaptor protein NCK-beta NCK2 GRB4 Growth factor receptor-bound protein 4 protein protein human Homo sapiens Human MTEEVIVIAKWDYTAQQDQELDIKKNERLWLLDDSKTWWRVRNAANRTGYVPSNYVERKNSLKKGSLVKNLKDTLGLGKTRRKTSARDASPTPSTDAEYPANGSGADRIYDLNIPAFVKFAYVAEREDELSLVKGSRVTVMEKCSDGWWRGSYNGQIGWFPSNYVLEEVDEAAAESPSFLSLRKGASLSNGQGSRVLHVVQTLYPFSSVTEEELNFEKGETMEVIEKPENDPEWWKCKNARGQVGLVPKNYVVVLSDGPALHPAHAPQISYTGPSSSGRFAGREWYYGNVTRHQAECALNERGVEGDFLIRDSESSPSDFSVSLKASGKNKHFKVQLVDNVYCIGQRRFHTMDELVEHYKKAPIFTSEHGEKLYLVRALQ g3hj13_crigr LIM and senescent cell antigen-like-containing domain protein I79_010647 protein protein crigr Cricetulus griseus (Chinese hamster) Cricetulus barabensis griseus Chinese hamster MANALASATCERCKGGFAPAEKIVNSNGELYHEQCFVCAQCFQQFPEGLFYEFEGRKYCEHDFQMLFAPCCHQCGEFIIGRVIKAMNNSWHPECFRCDLCQEVLADIGFVKNAGRHLCRPCHNREKARGLGKYICQKCHAIIDEQPLIFKNDPYHPDHFNCANCGKELTADARELKGELYCLPCHDKMGVPICGACRRPIEGRVVNAMGKQWHVEHFVCAKCEKPFLGHRHYERKGLAYCETHYNQLFGDVCFHCNRVIEGDVVSALNKAWCVSCFACSTCNTKLTLKNKFVEFDMKPVCKKCYEKFPLELKKRLKKLAETLGRK lims1-ilk-4 1390768 n/a 1390770 unspecified role unspecified role prey prey ank repeats sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false 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 ecoli Escherichia coli (strain K12) n/a 1390771 unspecified role unspecified role bait bait n_terminus 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 false lim domain required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) 1390780 1390776 direct interaction direct interaction false false false Fig 5. lims1-nck2-1 1390768 n/a 1390772 unspecified role unspecified role prey prey 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 ecoli Escherichia coli (strain K12) n/a 1390771 unspecified role unspecified role bait bait lim domain required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n_terminus 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 false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig 5. positive control ilk-lims1-2 1390768 n/a 1390771 unspecified role unspecified role prey prey n_terminus mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) n/a 1390770 unspecified role unspecified role bait bait ank1_2 required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false n_terminus 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 false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig 6A. The deletion of the first Ankyrin repeat from ILK [66-452] abolished the interaction between ILK and PINCH. ilk-g3hj13 1390769 n/a 1390773 unspecified role unspecified role prey prey n/a 1390770 unspecified role unspecified role bait bait n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ank repeats sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig 7B. Though the Ankyrin repeats region of ILK [1-230] was sufficient to interact with PINCH, the interaction was considerably less compared to wild type ILK.