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Transfection and generation of stable cell lines
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Wild-type DME cDNA and a catalytically inactive mutant version (D1562A) were subcloned into pcDNA3.1 vector (Invitrogen) from pET30b-DME and pET30b-DMED1562A, respectively [20], by KpnI/NotI digestion. DLD-1 cells were transfected with the empty plasmid or the different constructs using Lipofectamine LTX (Invitrogen), following the manufacturer´s recommendations. Stable transfectants were obtained after 2 weeks selection with 1 mg/ml G418 (Calbiochem).
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