Transfection of HEK293T cells

The day before transfection 0.2–0.4 × 10⁶ cells were seeded. On the day of transfection between 1.5 μg (for 1 well of a 12 well plate) and 2 μg (for 1 well of a 6 well plate) of DNA were diluted into DMEM serum free medium (100 μl for 1 well of a 12 well plate and 200 μl for 1 well of a 6 well plate). Polyethylenimine (PEI, Polysciences Inc. 23 966, Bergstrasse, Germany) was added to the DNA mix in a ratio DNA (μg): PEI (μg) of 1:3 and the DNA mix was incubated for 15 min. Fresh DMEM (10% v/v fetal calf serum, 5%v/v P/S) was added to the HEK293T cells and the DNA mix was added drop-wise to each well. The medium was refreshed after 4–6 h to prevent PEI toxicity. Forty-eight hours post-transfection cells were harvested. Cell pellets were taken up in 1X Laemmli sample buffer (0.2 M Tris–HCl [pH 6.8], 1.5% sodium dodecyl sulfate [SDS], 10% glycerol, 1 mm EDTA, 0.004% bromophenol blue) containing protease inhibitor cocktail and cells were lysed by sonication (3 × 4 s, 5 mAmp). Protein lysate (20 μg) was analyzed by SDS-PAGE and western blotting.