Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 3. Site 1 binds two distinct proteins and forms two DNA-protein complexes. A, sequences of the oligonucleotides used in Fig. 3 are shown. Only one strand is shown for each oligonucleotide. The numbering system uses translation start site as 11. B, an oligonucleotide spanning 268 to 239 of the Factor X gene (FXSite1) was labeled and incubated with 10 mg of nuclear extract (N.E.) from cell lines and rat tissues as indicated. Two DNA-protein complexes are designated complex A and complex B. The position of the free probe is marked as F. C, an oligonucleotide spanning site 1 was incubated with 12 mg of nuclear extract (N.E.) from human liver (lanes 1–12), 2 ml of in vitro translated HNF-4 in rabbit reticulocyte lysate (lane 13), or 2 ml of unprogrammed rabbit reticulocyte lysate (lane 14). Lanes 2–5 contain unlabeled site 1 oligonucleotide as cold competitors in 20 3, 100 3, 500 3, and 1000 3 molar excess. Lanes 7–10 contain unlabeled APF-1 (a strong HNF-4 binding site derived from 287 to 266 of apolipoprotein C III gene) as cold competitors in 20 3, 100 3, 500 3, and 1000 3 molar excess. Lane 12 contains 1 ml of HNF-4 antiserum. The positions of the antibody-HNF-4-DNA complexes are indicated by SS. D, probes containing the ACTTTG common motif from Factors VII, IX, X, and APF-1 were incubated with 12 mg of human liver nuclear extracts (N.E.) (lanes 1–8). One ml of HNF-4 antiserum was added in lanes 5–8. Positions of the immune complexes (supershift) are marked as SS. Two DNA-protein complexes specific to the APF-1 probe are marked by asterisks. E, the Factor X site 1 probe was incubated with 15 mg of nuclear extract (N.E.) from either HepG2 (lanes 1–3) or HeLa cells (lanes 4–6). In lanes 2 and 5, 200 3molar excess of unlabeled Sp1 consensus oligonucleotide was added. In lanes 3 and 6, 1 ml of Sp1 antibody was added. The position of a minor complex is denoted by an asterisk.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Labeling, Incubation, In Vitro, Binding Assay, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 4. Methylation interference analysis of the contact points of HNF-4 and Sp1 to site 1. A, On the left, Factor X site 1 was labeled on one strand (sense strand or antisense strand) and partially meth- ylated. The methylated probe was incubated with 100 mg of human liver nuclear extract and analyzed in regular electrophoretic mobility shift assay. The free probe fraction (F) and the complex B fraction (B, which contains HNF-4) were excised from the gel, cleaved with piperidine to reveal the G/A ladder, and resolved on a denaturing gel. On the right, the site 1 oligonucleotide was incubated with 10 footprint units of recombinant Sp1 and analyzed as described above. The free fraction (F) and bound fraction (B) were analyzed. The contact points revealed are marked as G. E indicates methylated positions that partially interfere with the binding. * indicates that methylation at these nucleotides enhances the protein binding. B, summary of results of methylation interference analysis at site 1.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Methylation, Labeling, Incubation, Electrophoretic Mobility Shift Assay, Recombinant, Binding Assay, Protein Binding

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 5. Increasing amounts of Sp1 diminish the binding of HNF-4 to site 1. Labeled Factor X site 1 oligonucleotide was incubated with 12 mg of human liver nuclear extracts (N.E.) in the presence of 0, 0.2, 1, and 2 footprint units (lanes 1–4) of recombinant Sp1.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Labeling, Incubation, Recombinant

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 1. Leptin receptor expression in glomerular endothelial cells (GER). (A) Ligand binding of [125I]-recombinant murine leptin to GER monolayers. Data are presented as Scatchard blot. Each point is the mean of four independent experiments performed in duplicate. Unspecific binding was performed in the presence of 1026 m nonradioactive murine leptin and is subtracted from the binding curve. GERs exhibit high-affinity receptors for leptin with a Kd of 4 nm and Bmax of 9700 receptors/cell. (B) Binding of 5 nm of [125I]-recombinant murine leptin in the presence of 5 mg of a goat polyclonal antimouse leptin receptor antibody (a-ObR Ab) or the same concentration of normal goat IgG. Total binding of leptin in the presence of normal goat IgG was considered as 100%. The a-ObR antibody, but not nonimmune goat IgG, significantly reduced binding of leptin to its putative receptor (P , 0.01, N 5 5 independent binding experiments). (C) Expression of the short rat isoform (r-Ob-Ra) but not the long form (r-Ob-Rb) in cultured GERs. cDNA amplification of reverse transcribed total RNA. A specific 487 bp band is detected in the r-Ob-Ra, but not the predicted 370 bp band in the r-Ob-Rb lane. This experiment was independently repeated five times with similar results. However, the r-Ob-Rb isoform could be easily amplified from rat whole brain cDNA using the same concentrations of primers and amplification conditions (data not shown).

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Expressing, Ligand Binding Assay, Recombinant, Binding Assay, Concentration Assay, Cell Culture, Reverse Transcription

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 4. Leptin stimulates expression of transforming growth factor-b1 (TGF-b1) in glomerular endothelial cells (GERs). (A) TGF-b1 protein secretion in the cell culture supernatant was also significantly stimulated by a single dose of leptin (6.25 to 625 nm) for 48 hours (N 5 3 to 4 independent stimulation experiments, *P , 0.05 vs. unstimulated controls). (B) Northern blot. A single dose of 0.62 to 625 nm leptin for 48 hours increased TGF-b1 mRNA expression. This blot is representative of three independent experiments with qualitatively similar results.

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Expressing, Cell Culture, Northern Blot

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 6. Glomerular mRNA expression in leptin-infused rats. (A) Intraperitoneal infusion of murine leptin with osmotic minipumps for 72 hours stimulated glomerular mRNA expression for PCNA and TGF-b1, but not for a1(IV) collagen. (B) Continuous infusion for three weeks increased glomerular transcripts for TGF-b1 and a1(IV) collagen, whereas PCNA mRNA returned to baseline. This blot is representative of three independent experiments with qualitatively similar results.

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Expressing

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 7. Immunohistological staining of renal sections from in vivo infusion experiments. (A and B) Staining of kidney sections of control or leptin- infused rats (72 hr) with a specific anti-PCNA antibody to assess in vivo proliferation of renal cells. Kidney section of rats infused with solvent for 72 hours revealed no glomerular PCNA staining, suggesting very low basal proliferation in normal glomeruli (A). In contrast, in leptin-infused animals PCNA-expressing cells are found in glomeruli (arrow; B). (C–F) Staining for collagen type IV in rats infused for three weeks with solvent or leptin. There was a light collagen type IV staining in mesangial matrix and tubular basal membranes of control-infused rats (C). However, an increase in glomerular collagen type IV staining was detected in rats infused with leptin for three weeks (D). A higher magnification reveals glomerular collagen type IV staining restricted to the mesangial area of control-infused animals (E), whereas leptin-infused animals exhibit a segmental increase in collagen type IV deposition (F). Magnifications 3150 for A–D, 3 300 for E and F.

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Staining, In Vivo, Control, Solvent, Expressing