cDNA of human or mouse GDE7 with a C-terminal FLAG tag was previously constructed16 (link). A cDNA sequence encoding a FLAG peptide was introduced by PCR using PrimeSTAR HS DNA Polymerase (TaKaRa Bio, Kusatsu, Japan) to construct cDNA for N-terminally FLAG-tagged GDE7. This was generated using a forward primer containing a XbaI site, Kozak sequence, FLAG, and 5×Gly linker, and a reverse primer with a BamHI site. Site-directed mutagenesis was performed by overlap extension PCR. Each DNA fragment was amplified using primer sets shown in Supplementary Table 1 and subcloned into the third generation lentiviral backbone vector containing a hygromycin resistant gene as previously described60 (link).
COS-7, MCF-7, and 3T3-L1 cells were transduced with each obtained lentivirus using 8 μg mL−1 hexadimethrine bromide (Sigma-Aldrich) and then selected with hygromycin B (Nacalai Tesque).
COS-7, MCF-7, and 3T3-L1 cells were transduced with each obtained lentivirus using 8 μg mL−1 hexadimethrine bromide (Sigma-Aldrich) and then selected with hygromycin B (Nacalai Tesque).
Free full text: Click here
