Figure 3

Identification of enhancer and repressor regions within the Smtnl1 promoter. In (A), various deletions (left) of the Smtnl1 promoter region spanning +100 bp to -2759 bp were used to drive expression of the Gaussia luciferase gene. The positions were numbered from the transcriptional start site (TSS) identified by 5'-RACE in this study. A7r5 cells were transiently co-transfected with the pGLuc reporter and the constitutively active β-galactosidase (β-gal) reporter vector, pbAct-β-gal for normalization. After 48 hr, the media and cell lysates were assayed for luciferase and β-gal activities, respectively. Luciferase activity was normalised to β-gal activity and expressed as fold-increase (right) relative to the activity of the empty pGLuc vector. All values are mean ± S.E.M. (n = 5-8). *- significantly different from +100 - 0 bp promoter region; #- significantly different from 0 to -118 bp promoter region. Statistical analysis was completed with ANOVA and Student-Neumann-Keuls post-hoc analysis, p < 0.05. Additional studies (B) also examined luciferase expression in Rat 1 fibroblast and human embryonic kidney (HEK 293) cell lines 48 hr after transient co-transfection with the pGLuc reporter vector and pbAct-β-gal for normalization. All values are mean ± S.E.M. (n = 3).