Figure 5. Mutation of C/EBPα site 2 results in the loss of UL84 binding in transfected cells.

HF cells (1 × 10⁷) were transfected by electroporation with either wt OriLyt (10μg) or pOriLyt-2/3 (10μg) and a UL84 (5μg) expression plasmid. Two days post transfection cells were prepared for ChIP assays using a UL84 specific antibody or, in the case of the control ChIP an unrelated antibody of the same isotype as the UL84 antibody. Lanes: 1, PCR product input DNA from cells cotransfected with wt OriLyt containing plasmid and a UL84 expression plasmid; 2, PCR product from a ChIP assay from cells transfected with an OriLyt containing plasmid and a UL84 expression plasmid; 3, PCR product from a ChIP assay from cells transfected with an OriLyt containing plasmid and a UL84 expression plasmid using an isotype control antibody. PCR reactions were performed using primers that flanked the C/EBPαβ transcription factor binding sites with in oriLyt: Forward 5′-ACTCGAGTCACCATCCCATAAT-3′ and reverse 5′-TTTTCGTAGAACGTTTCGTTAGAAG-3′. The plasmid used in the transfection experiments is shown at the left of the figure.