Journal: Diabetologia

Article Title: Regulation of human resistin gene expression in cell systems: an important role of stimulatory protein 1 interaction with a common promoter polymorphic site.

doi: 10.1007/s00125-005-1762-y

Figure Lengend Snippet: Fig. 1 Binding of Sp1 to the region around −420G of the resistin gene promoter. a Sche- matic diagram of a part of the human resistin gene, indicating a sequence variation at −420 of the resistin gene. b Electrophoretic mobility shift assays. Double- stranded oligonucleotides repre- senting the region from −433 to −406 bp of the resistin gene were radiolabelled with [α-32P]-dCTP and incubated without nuclear extracts (lanes 1 and 6), with nuclear extracts from 3T3-L1 adipocytes (lanes 2–5) or with nuclear extracts from THP-1 monocytes (lanes 7–10). Ap- proximately 1 μg of the antibod- ies against Sp1, Ikaros or KLF4 were used for the super-shift experiment. Three specific DNA–protein complexes (bands 1, 2 and 3) and free probes are indicated. The −420G/C varia- tion-independent band is indi- cated by an asterisk, and the fact that this band is not related to the −420G/C variation was proven in the previous work [21]

Article Snippet: The labelled probe (25,000 cpm) was incubated with nuclear proteins (5 μg) in 10 mmol/ HEPES, pH 7.9, containing 50 mmol/l KCl, 0.1 mmol/l EDTA, 0.25 mmol/l DTT, 0.1 mg/ml poly(dIdC), 0.01% Nonidet P-40, and 10% glycerol, at room temperature for 15 min. Antibodies against Sp1 (sc-17824), Sp3 (sc-644), Ikaros (sc-9861) or Kluppel-like factor 4 (KLF4) (sc-01905) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated with nuclear proteins for 15 min on ice before the addition of the labelled probe.

Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Incubation