Full length MRE11 was sub-cloned from pACT2 MRE11 plasmid clone (41 (link)), into pLXIN (to create pLXINWT) retroviral vector (Clontech) then the Quick Change Site-Directed Mutagenesis kit (Stratagene) was used to create the MRE11S676AS678A mutant (ATLDMUT). MRE11 cloned into pEYFP-C1 was kindly provided by Jean-Yves Masson (42 (link)), and the alanine MRE11S676AS678A mutant (non-phosphorylatable) and aspartic acid MRE11S676DS678D (phosphomimetic) mutants were made using site directed mutagenesis and sequence confirmed.
Plxin
Manufactured by Takara Bio
Sourced in United States
PLXIN is a laboratory equipment product offered by Takara Bio. It is a core functional device designed for specific laboratory applications. Further details on its intended use or applications are not available within the scope of this response.
Automatically generated - may contain errors
3 protocols using plxin
1
Generating MRE11 Mutant Constructs
2
Retroviral Expression Vectors for Kinases
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Expression vectors for hemagglutinin-tagged wild-type MEK1 (wtMEK) and caMEK, with a conversion of S218 and S222 RAF1-dependent regulatory phosphorylation sites to aspartic residues, were provided by Dr J. Pouysségur (Nice, France). The HA-wtMEK and HA-caMEK constructs were subcloned into the retroviral expression vector pLXIN (Clontech, Mountain View, CA, USA), as previously described.23 (link), 24 (link) The pRL-SV40 Renilla luciferase reporter vector was from Promega (Nepean, ON, Canada). Expression vectors encoding for human ΔNTCF4, wt LRP6, LRP6-5A and KRASG12V were all provided by Addgene (Cambridge, MA, USA). The TCF reporter constructs TOPFLASH and its negative control FOPFLASH as well as the c-myc promoter reporter (4 × TBE2) and its control (4 × TBE2-mutated) were also purchased from Addgene.
3
Optimized Mammalian Expression of XRS2
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The XRS2 ORF optimised for expression in mammalian cells using the GeneOptimizer algorithm [23 (link)] was synthesized and subcloned in an appropriate vector by Mr Gene (Regensburg, Germany). This construct was used for subcloning into the vector pCMV-Tag2b (Stratagene) allowing the expression of a Flag-tagged version of Xrs2 under control of the CMV promoter. Similarly, the XRS2 (WT) and NBN ORFs were also ligated into pCMV-Tag2b. All constructs were verified by direct sequencing. Enzymes SalI and NotI were used to cut the XRS2 and NBN ORFs from the pCMV-Tag2b constructs which were than treated with DNA PolymeraseI and ligated into the HpaI site of pLXIN (Clontech, USA, Mountain View, CA). The DNA sequence of all ORFs was verified by Sanger sequencing.
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