Transfection of HEK293T Cells

HEK293T cells were seeded in 24 well plates (Greiner Bio-One) at a concentration of 1 × 10⁵ cells per well on a coverslip for immunofluorescence experiments or at 2 × 10⁵ cells per well for secreted embryonic alkaline phosphatase assays. For immunoprecipitation, HEK293T cells were seeded in 10 cm cell culture dishes (Greiner Bio-One) at 4 × 10⁶ cells per dish. Cells were seeded 1 day before transfection. On the day of transfection, FuGENE 6 Transfection Reagent (Roche) was added into Opti-MEM® I (1x) in GlutaMAX(TM)-I (Gibco, Life Technologies) and incubated for 5 min at room temperature before mixed with relevant plasmids according to manufacturer's instructions. Transfection mixtures were incubated for 20 min before added to seeded cells. Transfected cells were placed in 37°C incubator with 5% CO₂ for 18 h incubation before harvesting for other experiments.