A, Chloramphenicol acetyltransferase activity expressed from a promoter transiently transfected into GHFT1-5 cells together with vectors expressing C/EBPα, fused or not, with GFP. The promoter contained a single C/EBPα binding site upstream of a TATA box . CAT activities were normalized to the activity present in cells transfected with the expression vector not containing the C/EBPα cDNA ("Sham") and plotted as the mean +/- standard deviation from five independent experiments. B, Nuclear extracts from sham-transfected cells and cells expressing C/EBPα, C/EBPα-GFP and GFP-C/EBPα were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a membrane and stained with an antibody directed against the FLAG epitope, which was appended to the amino terminus of C/EBPα in all the constructs. Arrow, expressed C/EBPα-GFP and GFP-C/EBPα. C, Whole cell extracts, from cells transfected with C/EBPα-GFP and GFP-C/EBPα or GFP-C/EBPαΔLZ expression vectors, were incubated with a radiolabeled oligonucleotide containing a high affinity consensus C/EBPα binding site. The observed complexes were competed with a 1, 10 and 100 fold molar excess of unlabeled oligonucleotide or were supershifted with an antibody directed against C/EBPα.