Localization and characterizaion of SRE in the GLUT2 promoter. A: Schematic diagram of serial deletion constructs of GLUT2 promoter reporter to localization of SRE in the GLUT2 promoter.
The indicated numbers represent the number of nucleotides from the ATG codon. B: Effect of SREBP-1c on the deletion constructs of GLUT2 promoter. The promoter activities were measured by cotransfecting
100 ng of SREBP-1c expression or empty vectors into Alexander cell lines. The results were normalized by the amount of total
protein of lysates, which were determined by the Bradford method (21) and shown as the fold changes of luciferase activities compared with those of the control. Normalized luciferase activities
are shown as the means ± SD of three independent experiments in triplicate. *P < 0.005 for the −166 vs. −57 deletion construct. C: Electrophoretic mobility shift assay of GLUT2-SRE. The assay was performed with recombinant SREBP-1 protein in 4% (wt/vol)
nondenaturing polyacrylamide gel. Fifty thousand cpm (0.1 pmol) of 32P-labeled GLUT2 promoter fragments (−101/−67) containing putative SRE were incubated with 20, 40, and 60 ng of recombinant
SREBP-1 protein. D: Effect of mutation on the SREBP-1 binding to the putative GLUT2-SRE. Site-directed mutation was introduced into the GLUT2-SRE
sequence. SRE mutant was prepared by replacing TGA with aac. Wild-type and mutant probes were labeled with [γ-32P]ATP, and EMSA was performed in 4% (wt/vol) nondenaturing polyacrylamide gel. For these experiments, 30 pmol of each double-stranded
oligonucleotide and 80 ng of SREBP-1 recombinant protein were used. Consensus sequence of SRE reported in the LDLR promoter
and mutant oligonucleotides were used as competitors. The DNA-protein complexes are indicated by an arrow. E: Effect of mutation on the GLUT2-SRE on the SREBP-1c–driven promoter activity. A pSV-SREBP-1c expression vector was cotransfected
with pmGT2d-389 or pmGT2d-389m into Alexander cell lines. The luciferase activities were represented as fold changes compared
with those of the control group. Values are the means ±SD of three independent experiments in triplicate.