HeLa cells or primary MEM cells prepared from Alx3-deficient embryos were seeded into 24-well plates at a density of 7 × 104 cells per well and incubated overnight. Cells were transfected for 4 hours with Lipofectamine (Invitrogen), using 500 ng of luciferase reporter plasmid, 50 ng of pRL-TK-Renilla and pcDNA3-Alx3 or control pcDNA3 plasmids. Luciferase and Renilla activities were measured using a commercial Dual Luciferase Assay kit (Promega, Madrid, Spain) 48 hours after transfection. The reporter plasmid RSV-Luc was used as an independent standard for normalization, and efficiencies were corrected by using the Renilla luciferase assay system (Promega). All transfections were carried out in duplicates.