Effect of artificial spacing between the adjacent Ets- and C/EBP-binding sites or mutation in the Ets-binding site on transactivation of the FCAR promoter by C/EBPα and C/EBPβ. (A) A schematic representation for the structure of the wild-type FCAR upstream-luciferase (Luc) construct (pGL-259) and the mutated FCAR upstream-luciferase constructs. The numbers indicate nucleotide positions relative to the translation initiation ATG at +1. The major transcription start site (-197) identified by de Wit et al⁴⁸  is indicated as bent arrow. The 3 C/EBP-binding sites at -139, -127, and -74 and an Ets-binding site at -92 are indicated. X indicates mutated site. (B-C) Jurkat cells were transfected with 0.7 μg luciferase reporter constructs containing the indicated FCAR promoter mutant in the absence or presence of 0.1 μg expression vector for C/EBPα (hCMV-C/EBPα) (B) or C/EBPβ (pD3NF-IL6) (C), along with 0.03 μg pRL-SV40; each sample was transfected with the additional corresponding empty vector pCMV-Empt (B) or pcDNA3.1(+) (C) to bring the amount of transfected DNA in each sample to 1 μg. Firefly and Renilla luciferase activities were determined 24 hours after transfection. Values are corrected for transfection efficiency and presented as the fold stimulation of each construct by C/EBPα (B) or C/EBPβ (C), compared with the promoter activity seen without the expression vector. Thin bars represent the SE from 3 to 4 transfections. ^*Significant decrease (P < .01) compared with pGLmCE12-259 by 2-tailed t test.