Transcriptional and DNA-binding activity in C/EBPα mutants.

C/EBPα mutants demonstrate severely impaired transcriptional and DNA-binding activity. (A) Western blot analysis of WT and mutant (F3901, J3, and F3820) C/EBPα proteins expressed in NIH3T3 cells from the pCMV-Sport1 vector. Similar expression levels for each protein were noted. (B) The pG-CSF receptor promoter–luciferase reporter construct (1 μg) was cotransfected with either empty (−), WT (50 ng), or mutant expression vector (100 ng). Relative firefly luciferase activity was measured and normalized to renilla luciferase activity. The fold change is indicated on the y-axis. The graph presents data from a duplicate experiment performed in triplicate. (C) Electrophoretic mobility shift assays (EMSAs) were performed with the use of the total cell lysates in panel A. An end-labled double-stranded oligonucleotide representing the C/EBP site in the G-CSF receptor promoter was incubated with the lysate in the absence (−) or presence (+) of 0.2 μg rabbit anti-C/EBPα antibody (Ab).