4.5. Transient Transfection

T-REx-293 cells were transfected with MaestroFectin transfection reagents (MaestroGen, Hsinchu City, Taiwan) as recommended by the manufacturer. Cells were seeded in 6 cm dishes and grown to 70–80% confluency before the transfection. The transfection procedure was conducted as follows: 3 μL MaestroFectin transfection reagent was first added to 90 μL serum-free medium (without antibiotics) in an Eppendorf tube and mixed gently. After incubating for 5 min at room temperature, 3 μg plasmid DNA was added to the diluted MaestroFectin transfection reagent and mixed gently. The mixture was incubated for 15 min at room temperature to form a MaestroFectin transfection reagent-DNA complex, and then 500 ng/mL tetracycline was added to the cells to induce the overexpression of target proteins for 18 h at 37 °C.