Fig. 3.

Characterization of Ric-8B gene promoter activity in osteoblastic cells. (A) Schematic diagram representing different segments of the Ric-8B gene promoter sequence evaluated through transient-transfection and luciferase activity assays. Gray ovals indicate putative C/EBP-binding sites. White boxes indicate CpG-rich regions, and black arrows represent different transcription start sites (TSS) described for this gene. Each of these promoter segments was cloned into the pGL3-Basic vector, upstream of the firefly luciferase gene. (B) Subconfluent MC3T3 cells were transiently transfected with increasing amounts (100 to 800 ng) of each of the constructs shown in panel A. Twenty-four hours later, luciferase activity was determined. p2.1Ric8B-Luc, p1.1Ric8B-Luc, p0.56Ric8B-Luc, and p0.33Ric8B-Luc correspond to constructs containing segments of the Ric-8B gene promoter of 2,124, 1,109, 558, and 327 bp, respectively. The bars represent the means ± standard errors of the means of two independent experiments, each performed in triplicate.