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We replicated these findings in three independent samples from Germany (KORA S4 and SHIP (Study of Health in Pomerania)) and Austria (SAPHIR; Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk), with P values ranging from 1.2 10.
After centrifugation, cell pellets were resuspended in 100 μl of 0.25 Tris-Cl, p H 7.8, and cells were lysed by three cycles of freeze-thawing.
The SLC2A9 genotypes also showed significant association with self-reported gout.
The proportion of the variance of serum uric acid concentrations explained by genotypes was about 1.2% in men and 6% in women, and the percentage accounted for by expression levels was 3.5% in men and 15% in women.
The gene encoding SHBG is also expressed in the Sertoli cell of the testis.
When the same transcription unit responsible for hepatic production of plasma SHBG is utilized in Sertoli cells, the protein product is most often referred to as the testicular androgen-binding protein.
transcription start site had the maximal activity among the constructs tested in Hep G2 cells (Fig.
3), we modified this promoter fragment to study the TTTAA sequence in FP1.After 10 min on ice, 25 fmol of end-labeled double-stranded oligonucleotide was added followed by a 15-min incubation at room temperature. Sladek) was diluted 1:5 in physiological saline containing 3% bovine serum albumin.For antibody supershift experiments, nuclear extracts were incubated (as above) on ice for 10 min before, and 15 min after, addition of the radiolabeled oligonucleotide. Tsai) was used undiluted and the HNF-4 antiserum (provided by Dr. Protein-DNA complexes were separated from the free probe by native 5% polyacrylamide gel electrophoresis in 0.45 All cell culture reagents were from Life Technologies, Inc. Human Hep G2 hepatoblastoma and He La cervical carcinoma cells were grown initially in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% fetal bovine serum, 2 m-activation experiments, the reporter plasmids (1 μg) were mixed with 100 ng of COUP-TF and/or HNF-4 expression plasmids and a p CMVlac Z control plasmid (200 ng).By contrast, a human transgene comprising the entire transcription unit encoding SHBG together with ∼0.9 kb of 5′-flanking sequence was expressed in the fetal2 and adult mouse livers, as well as in the adult mouse kidney (13). Radiolabeled probes were prepared by linearizing plasmids containing the wild type or mutated I and were purified by 6% polyacrylamide gel electrophoresis.Little, if any, expression of this human proximal promoter sequences that flank the transcription units for human SHBG in the liver and rat androgen-binding protein in the testis, and we used mouse liver nuclear protein extracts and human hepatoblastoma cells to identify possible targets for transcriptional regulators within the human promoter-luciferase reporter construct. DNase I (Amersham Pharmacia Biotech, FPLC pure) was used at a dilution (0.45 units/20-μl reaction) to digest approximately 20,000 cpm of end-labeled probe in an footprinting assay (16).All PCR-generated and mutated sequences were confirmed by DNA sequencing using a commercially available kit (Pharmacia Biotech, Uppsala, Sweden). The DNase I digestions were terminated, and the products were treated with proteinase K and analyzed by electrophoresis on an 8% polyacrylamide/urea DNA sequencing gel followed by exposure to an x-ray film.