pPICZ-mEGFP-hPORCN

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	The cells were transiently transfected using polyethyleneimine (PEI) [55]. The evening before the day of transfection, cells from T-75 flasks or 150 mm cell culture dishes were passaged and ~ 0.25 ×106 cells seeded into 12-well plates with 1 ml media per well. The next morning, 1 μg DNA was diluted into 50 μl of DMEM media. A separate tube contained 3 ug of PEI diluted into 50 μl of DMEM media.

Protocol tips
The cells were transiently transfected using polyethyleneimine (PEI) [55]. The evening before the day of transfection, cells from T-75 flasks or 150 mm cell culture dishes were passaged and ~ 0.25 ×106 cells seeded into 12-well plates with 1 ml media per well. The next morning, 1 μg DNA was diluted into 50 μl of DMEM media. A separate tube contained 3 ug of PEI diluted into 50 μl of DMEM media.

Publication protocol

HEK293T cells were maintained in DMEM media supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin/streptomycin in a humidified incubator at 37°C with 5% CO2. The cells were transiently transfected using polyethyleneimine (PEI) [55]. The evening before the day of transfection, cells from T-75 flasks or 150 mm cell culture dishes were passaged and ~ 0.25 ×106 cells seeded into 12-well plates with 1 ml media per well. The next morning, 1 μg DNA was diluted into 50 μl of DMEM media. A separate tube contained 3 ug of PEI diluted into 50 μl of DMEM media. The two solutions were mixed, pipetted, and incubated for 15–20 min at room temperature. Then ~ 100 μl per well of the DNA:PEI complex was added dropwise to the wells, the plate gently agitated and returned to the 37°C incubator. Alternatively, post addition of the DNA:PEI complex, the plate was incubated in a humidified shaker maintained at 30°C with 5% CO2 and the shaking turned off. For hPORCN constructs, we obtained more consistent results in doing the transient transfections in 6-well plates seeded with ~0.75×106 cells and transfected using 2 μg DNA complexed with 6 μg PEI per well. Larger scale transfections for protein purifications were done in 150-mm cell culture dishes. The dishes were initially seeded with ~10×106 cells and transfected the next day with 30 μg DNA complexed with 90 μg PEI in a final volume of 2 ml DMEM media.

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