2009
DOI: 10.1016/j.cellsig.2009.09.004
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Spatial and temporal activation of the small GTPases RhoA and Rac1 by the netrin-1 receptor UNC5a during neurite outgrowth

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Cited by 49 publications
(49 citation statements)
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References 61 publications
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“…Protein lysates were subjected to centrifugation at 10,000 ϫ g for 2 min at 4°C to remove insoluble materials. Endogenous GTP-Rac1 was pulled down by incubating the protein lysates for 30 min at 4°C with the Cdc42/Rac interactive binding domain (CRIB) of mouse PAK3 (amino acids 73 to 146) fused to GST and coupled to glutathione-Sepharose beads (19,42). The beads were washed twice with 25 mM HEPES (pH 7.5), 1% NP-40, 30 mM MgCl 2 , 40 mM NaCl, and 1 mM DTT and resuspended in SDS sample buffer.…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…Protein lysates were subjected to centrifugation at 10,000 ϫ g for 2 min at 4°C to remove insoluble materials. Endogenous GTP-Rac1 was pulled down by incubating the protein lysates for 30 min at 4°C with the Cdc42/Rac interactive binding domain (CRIB) of mouse PAK3 (amino acids 73 to 146) fused to GST and coupled to glutathione-Sepharose beads (19,42). The beads were washed twice with 25 mM HEPES (pH 7.5), 1% NP-40, 30 mM MgCl 2 , 40 mM NaCl, and 1 mM DTT and resuspended in SDS sample buffer.…”
Section: Methodsmentioning
confidence: 99%
“…In order to determine if the reduction in neurite outgrowth by Trio Y2622F was due to an impaired GEF activity toward Rac1, we performed Rac1 pulldown assays to assess the levels of activated, endogenous GTP-Rac1 trapped by specific binding to the CRIB domain of Pak fused to GST (31,42). As expected, Trio expression in HEK293 cells increased Rac1-GTP levels ( Fig.…”
Section: Phosphorylation Of Trio Downstream Of the Src Family Kinase mentioning
confidence: 99%
“…We have analysed loss-of-function mutants for Drosophila homologues of these possible downstream molecules, but none showed phenotypes comparable to those of fra 3 /fra 4 or unc5 8 (data not shown). Previous data demonstrate a function for the small GTPases Rac1 and Rho1 in ePG migration in Drosophila (Sepp and Auld, 2003), and it was recently shown that they can act downstream of Unc5 signalling in vertebrates (Picard et al, 2009). We expressed a dominant-negative form of Rho1 using cas-Gal4 in an otherwise wild-type background and obtained stalling of ePG6 and ePG8 (cas>Rho1N19, data not shown).…”
Section: Selectivity Of Netrin-mediated Signalling In Glial Cell Migrmentioning
confidence: 99%
“…The YPet sequence was amplified from pCEP4YPet-MAMM (Addgene plasmid 14032) and cloned into AgeI/NotI sites of pEYFP-N1 (Clontech), generating the pYPet-N1 (Nguyen and Daugherty 2005). The pECFP-RhoA construct was previously described (Picard et al 2009). …”
Section: Plasmids and Antibodiesmentioning
confidence: 99%