PMC:4829102 / 23614-25515 JSONTXT

{"project":"2_test","denotations":[{"id":"26807646-20541610-73828205","span":{"begin":1558,"end":1560},"obj":"20541610"},{"id":"26807646-20541610-73828205","span":{"begin":1558,"end":1560},"obj":"20541610"}],"text":"Plasmids\nThe open reading frame (ORF) of human EXD2 was purchased as a gateway entry clone in the pDONR221 plasmid backbone from DNASU Plasmid Repository (HsCD00295838). Discrepancies in the amino-acid sequence in comparison to the reference sequence for human EXD2 (NM_001193360.1) were corrected by site-directed mutagenesis. Site-directed mutagenesis was then employed to generate EXD2 D108A–E110A in pDONR221.\nFlag–HA–EXD2 WT and D108A–E110A as well as GST–EXD2 WT and D108A–E110A plasmid constructs were generated by recombination of the WT or D108A–E110A EXD2 ORF in pDONR221 by LR Clonase reaction into either the pHAGE-N-Flag–HA destination vector (a gift from R. Chapman, The Wellcome Trust Centre for Human Genetics, University of Oxford, UK), or the pDEST-pGEX6P-1 destination vector (a gift from C. Green, The Wellcome Trust Centre for Human Genetics, University of Oxford, UK), respectively. LR Clonase reactions were carried out using the Gateway LR Clonase II enzyme mix according to the instructions of the manufacturer (Life Technologies). The pCMV-I-Sce1 plasmid was a gift from V. Macaulay (Department of Oncology, University of Oxford, UK). pmCherry-C1 was obtained from Clontech. pX335-GFP plasmid (pX335 vector48 containing PGK-EGFP-P2A-Neo-pA) was a gift from J. Riepsaame and M. de Bruijn (MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK).\nHis–EXD2 (Lys76–Ser589) construct was generated by cloning of truncated human EXD2 (Lys76–Ser589) in the expression vector pNIC28-Bsa4 (ref. 49), containing an amino-terminal His tag followed by a tobacco etch virus protease cleavage site. The construct was subsequently subjected to site-directed mutagenesis to introduce the D108A–E110A mutations. Plasmids were transfected into human cells using Lipofectamine 2000 (Life Technologies), according to the manufacturer’s instructions."}