Figure 1.

Binding of C/EBPα to the Lactoferrin (LF) promoter and expression of the LF gene during neutrophil maturation in ATRA-induced MPRO cells. A. Northern blot analysis of uninduced and ATRA-induced MPRO cells demonstrating an increase in LF mRNA expression upon ATRA-treatment of cells for 24 and 48 hours. Ethidium bromidestained 28S rRNA in the lower panel serves as a loading control. B. Oligonucleotide pull down assay using LF C/EBP oligomers and extracts prepared from HEK293T cells. Nuclear extracts prepared from HEK293T cells overexpressing a C/EBPα expression plasmid were incubated with biotinylated wildtype LF/C/EBP oligomers. The DNA-protein complexes were recovered using streptavidin–agarose beads and the bound proteins resolved by SDS-PAGE and western blot analysis. The blot was probed with a C/EBPα antibody (lanes 1 and 2). 10% of the input sample is represented in lane 3 (In). C. Oligonucleotide pulldown assay using LF/ C/EBP oligomers and nuclear extracts from Uninduced and induced MPRO cells. Nuclear extracts prepared from uninduced (0) and ATRA-induced (48h) MPRO cells (a murine myeloid cell line for normal neutrophil maturation) were incubated with wild type (WT) or mutant (mut) biotinylated C/EBP site (LF/C/EBP) oligomers from the LF promoter. The DNA-protein complexes were recovered using streptavidin–agarose beads and the bound proteins resolved by SDS-PAGE and western blot analysis. The blot was probed sequentially with C/EBPα and C/EBPε antibodies.