sp1 consensus sequence probe  (Promega)

Journal: The American Journal of Pathology

Article Title: High Glucose–Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase

doi: 10.1016/j.ajpath.2016.12.011

Figure Lengend Snippet: Specificity of specificity protein (Sp)-1–binding with human MIOX gene and modulation of its promoter activity. A: The specificity of binding of differentially methylated segment (DMS)-1 oligo was assessed by using anti–Sp-1 antibody in eletrophoretic mobility shift assays. A distinct band is seen when nuclear extracts are incubated with DMS1 (arrowhead). On subsequent incubation with the antibody, the band shifts to higher position (supershift), suggesting the formation of a DMS1-DNA:nuclear protein:Sp-1 antibody complex, and binding seems to be specific (big arrow). B: Validation of Sp-1 binding was assessed with the use of the Sp1 gene–specific inhibitor mithramycin. An increase in the intensity of the band is observed with the incubation of DMS1 oligo under high-glucose (30 mmol/L) ambience (arrowhead). Co-treatment of HK-2 cells with 200 nm of mithramycin notably reduces the intensity of the band under high-glucose ambience, whereas its intensity is drastically reduced under low glucose (5 mmol/L) ambience. C: Whether Sp-1 modulates the MIOX promoter activity was assessed by mutational analyses, where mutant reporter constructs were generated. Under low-glucose ambience, a mild relative luciferase (LUC) activity is observed, and it is notably reduced with a single base pair mutation (C → T) within the MIOX promoter. Under high-glucose ambience, a remarkable increase in the promoter activity is observed, and it is markedly reduced with the mutation in the Sp-1–binding site. The asterisks and small arrows in A and B indicate nonspecific bands. Data are expressed as means ± SEM. n = 4 independent experiments (C). ∗P < 0.05 versus the respective control. CDS, coding segment.

Article Snippet: Double-stranded probe with Sp1 consensus sequence was purchased from Santa Cruz (catalog number sc-2502).

Techniques: Binding Assay, Activity Assay, Methylation, Mobility Shift, Incubation, Biomarker Discovery, Mutagenesis, Construct, Generated, Luciferase, Control

Journal: The American Journal of Pathology

Article Title: High Glucose–Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase

doi: 10.1016/j.ajpath.2016.12.011

Figure Lengend Snippet: Effect of Sp1 gene disruption on the expression of specificity protein (Sp)-1, myo-inositol oxygenase (MIOX), NADPH oxidase (NOX)-4, hypoxia-inducible factor (HIF)-1α, and fibronectin (Fn) under high-glucose ambience. Under high-glucose (30 mmol/L) ambience, Sp-1 and MIOX expression increases compared with the control low-glucose (5 mmol/L) ambience and is reduced by the Sp1 siRNA treatment (A, B, F, G, and K). The other relevant parameters, such as tubular NOX-4 and HIF-1α, increase under high-glucose ambience and decrease after Sp1 siRNA treatment (C, D, H, I, and K). Fibronectin was used as a distal readout. Fibronectin cellular mRNA and protein expression increases under high-glucose ambience and reduces with the treatment of Sp1 siRNA (E, J, and K). The scramble oligo has no significant effect in any of the readouts. Data are expressed as means ± SEM. n = 4 independent experiments (A–J). ∗P < 0.05 versus the respective control.

Article Snippet: Double-stranded probe with Sp1 consensus sequence was purchased from Santa Cruz (catalog number sc-2502).

Techniques: Disruption, Expressing, Control

Journal: The American Journal of Pathology

Article Title: High Glucose–Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase

doi: 10.1016/j.ajpath.2016.12.011

Figure Lengend Snippet: Effect of Sp1 gene disruption on the cellular morphologic findings related to the expression of myo-inositol oxygenase (MIOX), specificity protein (Sp)-1, and fibronectin (Fn) under high-glucose ambience. B and D: The expression of MIOX markedly increases in the cytoplasm of HK-2 cells under high-glucose (HG) ambience and reduces after Sp1 siRNA treatment. A and C: A mild decrease is also observed after Sp1 siRNA treatment in cells subjected to low-glucose (LG) ambience. E and F: The expression of Sp-1 confines to the nuclear compartment and increases considerably under HG ambience in the hypertrophic cells with enlarged nuclei. G and H: Decrease in Sp-1 expression after siRNA treatment in cotreatment with LG and HG. I–L: The Fn expression markedly increases under HG environment (I and J) and reduces after Sp1 siRNA treatment (L). I and K: No difference in the Fn expression is observed in cells maintained under basal LG environment and treated with Sp1 siRNA. Scale bars = 40 μm (A–L).

Article Snippet: Double-stranded probe with Sp1 consensus sequence was purchased from Santa Cruz (catalog number sc-2502).

Techniques: Disruption, Expressing

Journal: The American Journal of Pathology

Article Title: High Glucose–Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase

doi: 10.1016/j.ajpath.2016.12.011

Figure Lengend Snippet: Effect of Sp1 siRNA on high-glucose–induced perturbations in mitochondrial redox in renal tubular cells. Compared with the control, an increased staining of dihydroethidium (DHE) is observed under high-glucose ambience, and this increase is also reflected in flow cytometric analyses (A, B, F, and G). Sp1 siRNA treatment considerably reduces the staining and the mitochondrial oxidant stress (B, C, G, and H). No significant differences in the DHE staining or generation of reactive oxygen species are observed in cells maintained under low-glucose ambience and treated with Sp1 siRNA (A, D, E, F, and I). Data are expressed as means ± SEM (E). n = 4 independent experiments (E). ∗P < 0.05 versus the respective control. Scale bars = 80 μm (F–I). Glu, glucose; S.Oligo, scramble oligo.

Article Snippet: Double-stranded probe with Sp1 consensus sequence was purchased from Santa Cruz (catalog number sc-2502).

Techniques: Control, Staining

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications

doi: 10.1111/j.1600-0897.2010.00854.x

Figure Lengend Snippet: EMSA with nuclear extracts from primary trophoblasts and THP-1 cells using probes corresponding to sequences encompassing −1082 (G or A), −592 (C or A), or Sp1 consensus motif. Three panels, free probe, primary trophoblast, and THP-1 represent the −1082 (lanes 1 and 2, 6 and 7, 11 and 12), the −592 (lanes 3 and 4, 8 and 9, 13 and 14), and the Sp1 consensus (lanes 5, 10, 15) sequences. The −1082 and −592 lanes represent G (lanes 1, 6, 11) or A (lanes 2, 7, 12) and C (lanes 3, 8, 13) or A (lanes 4, 9, 14) genotypes, respectively. The free probe is not visible because the panels only represent DNA–protein complexes. Note the EMSA differences between primary trophoblasts and THP-1 cells. There are two critical bands that represent specific DNA–protein complexes. Nuclear extract from primary term cytotrophoblasts, the faster migrating complex is prominent, whereas the slower migrating complex is prominent with nuclear extract from THP1 cells. The −592 sequences are the binding site with nuclear extract from trophoblast cells, whereas THP1 nuclear extract gives a similar profile with both the −1082 and −592 sequences. The DNA–protein complex profile with the consensus Sp1 binding sequences also differs between primary trophoblasts and THP1 cells.

Article Snippet: Nuclear protein extracts from primary placental cells and THP-1 cells were also tested with radiolabeled Sp1 consensus sequence (Promega, Madison, WI, USA).

Techniques: Binding Assay

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications

doi: 10.1111/j.1600-0897.2010.00854.x

Figure Lengend Snippet: The −1082 site DNA–protein complex does not involve participation of Sp1 in primary trophoblasts as demonstrated by lack of supershift by Sp1 antibody. EMSA and supershift were performed as described in Materials and Methods. Both the −1082 A (lanes 1–4) and −1082 G (lanes 5–8) probes were used. Lanes 1 and 5 represent free probes. Nuclear extracts were isolated from LPS untreated and treated primary trophoblasts. Lanes 2 and 6 represent nuclear extract from untreated cells, whereas lanes 3 and 7 contained nuclear extract from LPS-treated cells. Supershift with Sp1 antibody was performed with nuclear extract from LPS-treated cells (lanes 4 and 8). Note no supershifted bands were observed in the presence of Sp1 antibody.

Article Snippet: Nuclear protein extracts from primary placental cells and THP-1 cells were also tested with radiolabeled Sp1 consensus sequence (Promega, Madison, WI, USA).

Techniques: Isolation

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications

doi: 10.1111/j.1600-0897.2010.00854.x

Figure Lengend Snippet: LPS treatment of primary trophoblasts induces Sp1 involvement in DNA–protein complexes at the −592A sequences, not at the −592C sequences. EMSA and supershift were performed as described in legend to Fig, 4. Panels a and b represent EMSA with −592A and −592C probes (see Methods and Materials). Lanes 1–5 represent free probes (lane 1), nuclear extract from untreated cells (lane 2), nuclear extract from LPS-treated cells (lane 3), nuclear extract LPS + 1 μL of Sp1 antibody (lane 4), and nuclear extract LPS + 5 μL of Sp1 antibody. Note that Sp1 antibody abolished the faster moving band observed with the −592A probe.

Article Snippet: Nuclear protein extracts from primary placental cells and THP-1 cells were also tested with radiolabeled Sp1 consensus sequence (Promega, Madison, WI, USA).

Techniques: