FIGURE 3.

Effect of wild-type and chimeric C/EBP proteins on the G-CSFR promoter. A, a histogram shows luciferase activity in 293T cells co-transfected with pTK-G-CSFR-luciferase and C/EBP plasmids or the MigRI empty vector after treatment (12 h) with 4-HT. Results (three independent experiments) are expressed as -fold activation over that in empty vector-transfected cells after normalization for Renilla luciferase activity and are reported as the means of triplicate determinations; error bars represent the S.D. of the mean; p values indicate statistical significance of the difference in transactivation activity of C/EBPα-ER versus C/EBPβ-ER or N-C/EBPβ-C/EBPα-DBD-ER calculated using unpaired, two-tailed Student's t test. B, quantitative ChIP assays show interaction of C/EBPα, C/EBPβ, and C/EBPα-C/EBPβ chimeric proteins with a segment of the G-CSFR promoter (nucleotides −147 to + 25) containing a functional C/EBPα binding site detected by real time Q-PCR. Error bars denote S.D. of the means of one representative experiment (of two) performed in triplicate. A Western blot shows total and immunoprecipitated (IP) C/EBPα, C/EBPβ, and chimeric proteins from K562 cell lysates used for ChIP assays. Chimeric proteins were immunoprecipitated and detected by Western blot with the anti-estrogen receptor α monoclonal antibody (Ab) (Stressgen).