Reporter assay for murine TfR2 promoter.

NIH-3T3 cells were transfected by Geneporter system (Gene Therapy Systems) with either empty pGL3-basic (C) or mTfR2 promoter reporter plasmids (P) pGL-mTfR2-pro-0.3 kb, -0.4 kb, -1.0 kb, or -2.1 kb together with expression plasmids for GATA-1 (G), FOG-1 (F), EKLF (E), and/or C/EBP-α (A). To normalize the transfection efficiency, pCMV  ·  SPORT-β-Gal (Life Technologies) was cotransfected. Cells were harvested after 60 hours, and luciferase and β-galactosidase activities were measured with a luminometer and with the β-galactosidase Enzyme Assay System (Promega), respectively. Experiments were performed in triplicate and were repeated twice, with very similar results. Mean values of relative luciferase activities and SDs of a representative experiment are shown.