mbinfo MBInfo http://www.mechanobio.info/ http://www.mechanobio.info/ del-2002-1 Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Del Pozo M.A., Kiosses W.B., Alderson N.B., Meller N., Hahn K.M., Schwartz M.A. Interaction detection: GST pull down. Since the specific RhoGDI used could not be identified or traced from references, interactions involving RhoGDI could not be curated. mdelpozo@scripps.edu, schwartz@scripps.edu Nat. Cell Biol. (1465-7392) 2002 Only protein-protein interactions imex curation del-2002-2 Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. mouse-nih_3t3 Mus musculus Swiss NIH 3t3 embryo fibroblast cells nih_3t3 Mouse Swiss NIH embryo contact inhibited fibroblast cells fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence Del Pozo M.A., Kiosses W.B., Alderson N.B., Meller N., Hahn K.M., Schwartz M.A. Interaction detection: GST pull down. Since the specific RhoGDI used could not be identified or traced from references, interactions involving RhoGDI could not be curated. mdelpozo@scripps.edu, schwartz@scripps.edu Nat. Cell Biol. (1465-7392) 2002 Only protein-protein interactions imex curation pak3_human Serine/threonine-protein kinase PAK 3 PAK3 OPHN3 Beta-PAK Oligophrenin-3 p21-activated kinase 3 protein protein human Homo sapiens Human MSDGLDNEEKPPAPPLRMNSNNRDSSALNHSSKPLPMAPEEKNKKARLRSIFPGGGDKTNKKKEKERPEISLPSDFEHTIHVGFDAVTGEFTPDLYGSQMCPGKLPEGIPEQWARLLQTSNITKLEQKKNPQAVLDVLKFYDSKETVNNQKYMSFTSGDKSAHGYIAAHPSSTKTASEPPLAPPVSEEEDEEEEEEEDENEPPPVIAPRPEHTKSIYTRSVVESIASPAVPNKEVTPPSAENANSSTLYRNTDRQRKKSKMTDEEILEKLRSIVSVGDPKKKYTRFEKIGQGASGTVYTALDIATGQEVAIKQMNLQQQPKKELIINEILVMRENKNPNIVNYLDSYLVGDELWVVMEYLAGGSLTDVVTETCMDEGQIAAVCRECLQALDFLHSNQVIHRDIKSDNILLGMDGSVKLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRKAYGPKVDIWSLGIMAIEMVEGEPPYLNENPLRALYLIATNGTPELQNPERLSAVFRDFLNRCLEMDVDRRGSAKELLQHPFLKLAKPLSSLTPLIIAAKEAIKNSSR rac1_human Ras-related C3 botulinum toxin substrate 1 RAC1 MIG5 p21-Rac1 Ras-like protein TC25 Cell migration-inducing gene 5 protein TC25 protein protein human Homo sapiens Human MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHHCPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRGLKTVFDEAIRAVLCPPPVKKRKRKCLLL pak3-rac1 1316672 n/a 1316674 unspecified role unspecified role bait bait p21-binding domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 false ecoli Escherichia coli (strain K12) n/a 1316675 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association false false false Fig 5a. The interaction was inhibited in the presence of increasing concentrations of Rho-GDI. pak3-rac1-2 1316673 n/a 1316674 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region alexa 546 label alexa 546 label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false p21-binding domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 1316675 unspecified role unspecified role fluorescence donor fluorescence donor region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P63000:p.Gly12Val mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false proximity proximity close-contact colocalization neighbourhood interaction false false false Fig 4c, 3a, 4a, 4b. The FRET signal was increased when Rac -G12V was used (Fig 3a).The FRET signal was increased when the cells microinjected with the two proteins were incubated with fibronectin-coated beads. (Fig 4a, 4c). Incubation with anti-beta1 integrin-coated beads also increased the FRET signal (Fig 4b). The FRET signal was decreased when the cells were incubated with anti-CD44-coated beads. The FRET signal was weak when the cells were injected with GFP-V12-Rac-6Q, even when incubated with fibronectin-coated beads (Fig 4d). The effect of the GFP-T35S/Q61L-Rac and GFP-G12V/C189S-Rac constructs on the FRET signal could not be curated since the source of these constructs could not be traced.