Fig. 4.

oNpt2a promoter activity in NHERF-1 deficient OK-H cells. Luciferase -reporter gene assays were performed in OK opossum kidney cells (OK-WT) and the NHERF-1-deficient OK-H cells. The cells were transiently transfected with pGL3 luciferase reporter gene constructs containing either 4.7 kb (p4700), 208 bp (p208), or 121 bp (p121) of the 5’-flanking region of opossum Npt2a gene [6]. The pGL3 basic reporter construct was used as negative controls. 24 hours post-transfection cell lysates were collected and luciferase activity determined and normalized to protein concentration. Triplicate transfections were performed for each experiment and the average RLU/μg protein values determined. A single experiment represents cells grown from independent cell stocks and shown are the mean RLU/μg protein values ± SE from 3 independent experiments. A confirmatory experiment using β-galactosidase as a transfection control was performed for normalization and the results were consistent with the data shown. The ratio of OK-H/ OK-WT RLU/μg protein values was determined for each construct to assess the activity in OK-H cells relative to the OK-WT cells. Shown in the inset are the mean relative values ± SEM from the 3 independent experiments. One-way ANOVA followed by Tukey’s post-hoc test was used to determine significant difference in activity. *, p < 0.001 compared to p4700 WT; +, p <0.01 compared to p208 WT.