|Always bring FuGENE HD to room temperature and mix well prior to use.
Cells at 50% confluency were transiently transfected using FuGENE HD Transfection Reagent (Roche) following the instruction of the manufacturer. Full length human cDNA subcloned into pCMV6-XL5 was purchased from Origene. Plasmid DNA was first diluted in OPTI-MEM I (Gibco-BRL, USA) and then mixed with FuGENE HD Transfection Reagent. Following a 20-minute incubation period at room temperature, the DNA-FuGENE HD Transfection Reagent mixture was added to the antibiotic-free transfection medium (DMEM containing glutamine). The cells were maintained in a 37°C incubator with a saturated humidity air atmosphere containing 5% CO. The medium was replaced with normal culture medium 6 hours later. Cells were used for further experiments 48 hours after transfection. Full paper
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