Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 2. Regulation of Smad signaling response to TGF- by BMP-7. HK-2 cells were transfected with Smad3-responsive (A and B) or Smad2-responsive (C) plasmids and then were incubated with BMP-7 for various times or doses, before incubation with 1 ng/ml TGF- (black bar, control medium followed by TGF-; gray bars, BMP-7 followed by TGF-) or control medium (white bars) for 6 hours. A: Effect on Smad3 signaling of time course of 50 ng/ml BMP-7 incubation. B: Effect on Smad3 signaling of 24 hours preincubation with a BMP-7 dose range of 0 to 2000 ng/ml. C: Effect on Smad2 signaling of a 24-hour preincubation with BMP-7 at a dose range of 0 to 2000 ng/ml. *P 0.05 versus TGF-. RLU, relative light units.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Transfection, Incubation, Control

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 3. Time course of Smad phosphorylation and dephosphorylation/ degradation in response to TGF- and BMP-7. A: HK-2 cells were incubated with 50 ng/ml BMP-7 for 0 to 24 hours before incubation with 1 ng/ml TGF- for 1 hour. Whole-cell lysates were immunoblotted with antibodies against Phospho-Smads (PSmad) 1, 2, 3, and total Smads 1 and 3. Stripping and reprobing for glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used to confirm approximately equal loading. B–D: Time course of Smad1/3 dephosphorylation/degradation. HK-2 cells were incubated with control medium (B and C) or 50 ng/ml BMP-7 (D) for 24 hours before incubation with 1 ng/ml TGF- for 30 minutes. Cells were washed extensively and incubated in cytokine-free control medium (B) or medium containing the Alk5 kinase inhibitor SB431542 (C and D) for time points up to 5 hours. Residual Phospho-Smad3 activity was detected by immunoblot before strip- ping and reprobing for total Smad3.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Incubation, Stripping Membranes, Control, Activity Assay, Western Blot

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 4. Nuclear accumulation of Smad3. HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours before incubation with 1 ng/ml TGF- or control medium for 1 hour. A: Immunoblotting of nuclear extracts for Phospho-Smad (PSmad) 1/3 and subsequent reprobing for c-Jun to confirm approximately equal loading. B: Immunofluorescent localization of Smad3. HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours in eight-well chamber slides before incubation with 1 ng/ml TGF- or control medium for 1 hour and detection of Smad3 by immunofluorescence microscopy.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Incubation, Control, Western Blot, Immunofluorescence, Microscopy

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 5. BMP-7 inhibits Smad3 DNA binding. A and B: Electrophoretic mobility shift assay with a consensus Smad binding element probe. HK-2 cells were incubated with BMP-7 or control (Ctrl) medium for 24 hours before incubation with 1 ng/ml TGF- or control medium for 1 hour. Bold arrow, retarded probe; arrow, supershifted probe. A: Electrophoretic mo- bility shift assay performed with nuclear protein extract and consensus Smad binding element (SBE) probe. B: Supershift assay performed with antibodies to Smad3, 4, and 5. Sm, Smad. C and D: ChIP: Smad3 binding to the PAI-1 promoter. After chromatin immunoprecipitation with Smad3 antibody or pre-immune globulin, PAI-1 promoter Smad binding elements (SBE) were detected by qRT-PCR. Data are presented as Smad3-precipitated signal/pre- immune globulin-precipitated signal, normalized to control. C: HK-2 cells were incubated with TGF- for time points to 24 hours before ChIP. D: HK-2 cells were incubated with BMP-7 and TGF- as indicated for 6 hours before ChIP. RE, Relative Expression.

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Control, Shift Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing

Journal: The American journal of pathology

Article Title: Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

doi: 10.2353/ajpath.2010.090459

Figure Lengend Snippet: Figure 8. Proposed mechanism of regulation of Smad3 signaling by BMP7. A: In the absence of TGF-, Smads shuttle into and out of the nucleus. SnoN binds to Smad binding elements and prevents R-Smad binding. B: After ligand binding, active TGF- receptor complex leads to R-Smad phosphorylation. R-Smad-Arkadia-SnoN complexes lead to SnoN degradation. R-Smad-Smad4 complexes accumulate in the nucleus and bind to DNA. C: BMP7 prevents loss of SnoN expression. R-Smad-Smad4 com- plexes accumulate in the nucleus, but DNA binding to consensus Smad binding elements is prevented by SnoN. Arkadia-dependent SnoN degrada- tion appears necessary for Smad3 (or Smad2 -exon3)-dependent responses, but not Smad1/Smad4-dependent responses or responses driven by Smad2- Smad4-FoxH1 complexes (see Levy et al26).

Article Snippet: Smad Binding Element Oligonucleotide Probe (sc-2603) was bought from Santa Cruz Biotechnology, Inc. and annealed for use in the electrophoretic mobility shift assay.

Techniques: Binding Assay, Ligand Binding Assay, Phospho-proteomics, Expressing

Journal: International journal of molecular medicine

Article Title: Sphingosine-1-phosphate ameliorates the cardiac hypertrophic response through inhibiting the activity of histone deacetylase-2.

doi: 10.3892/ijmm.2017.3325

Figure Lengend Snippet: Figure 1. Effects of siRNAs targeting S1PR1‑3 in H9c2 cells. (A) Representative immunoblots for S1PR1, S1PR2 and S1PR3 and (B) semi‑quantification of S1PRs protein expression in H9c2 cells. Data are presented as the means ± standard error of the mean (n≥3 for each experiment). *P<0.05 vs. siNC‑transfected cells. S1PR, sphingosine‑1‑phosphate receptor; siNC, negative control siRNA; siR1‑3, siRNA‑S1PR1‑3; siRNA, small interfering RNA.

Article Snippet: Antibodies against atrial natriuretic peptide (ANP; sc-20158), brain natriuretic peptide (BNP; sc-271185), S1PR2 (sc-25491) and GAPDH (sc-32233) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Western Blot, Expressing, Negative Control, Small Interfering RNA

Journal: International journal of molecular medicine

Article Title: Sphingosine-1-phosphate ameliorates the cardiac hypertrophic response through inhibiting the activity of histone deacetylase-2.

doi: 10.3892/ijmm.2017.3325

Figure Lengend Snippet: Figure 6. S1PR expression is altered following TAC; S1PR2 may be involved in the antihypertrophic effects of S1P. (A and B) Representative immunoblots and semi‑quantification of S1PRs in the hearts of mice in the sham and TAC groups. Data are presented as the means ± standard error of the mean (n≥5 for each group). *P<0.05 vs. the Sham group. (C‑H) Representative immunoblots and semi‑quantification of β‑MHC in H9c2 cells following various treatments Data are presented as the means ± standard error of the mean (n≥3 for each experiment). *P<0.05 vs. Con group; #P<0.05 vs. PE group; &P<0.05 vs. PE + S1P group. β‑MHC, β‑myosin heavy chain; Con, control; PE, phenylephrine; S1P, sphingosine‑1‑phosphate; siNC, negative control siRNA; siR1‑3, siRNA‑S1P receptors 1‑3; siRNA, small interfering RNA; TAC, transverse aortic constriction.

Article Snippet: Antibodies against atrial natriuretic peptide (ANP; sc-20158), brain natriuretic peptide (BNP; sc-271185), S1PR2 (sc-25491) and GAPDH (sc-32233) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control, Negative Control, Small Interfering RNA

Journal: International journal of molecular medicine

Article Title: Sphingosine-1-phosphate ameliorates the cardiac hypertrophic response through inhibiting the activity of histone deacetylase-2.

doi: 10.3892/ijmm.2017.3325

Figure Lengend Snippet: Figure 7. Suppressive effects of S1P on HDAC2 activity are independent of S1PR2. (A) Nuclear HDAC2 activity was determined in H9c2 cells following various treatments. (B and C) Representative immunoblots and semi‑quantification of KLF4 expression in H9c2 cells following various treatments. Data are presented as the means ± standard error of the mean (n≥3 for each experiment). *P<0.05 vs. Con group; #P<0.05 vs. PE group; ns, not significant. Con, control; HDAC2, histone deacetylase‑2; KLF4, Krüppel‑like factor 4; PE, phenylephrine; S1P, sphingosine‑1‑phosphate; siNC, negative control siRNA; siR2, siRNA‑S1PR2; siRNA, small interfering RNA

Article Snippet: Antibodies against atrial natriuretic peptide (ANP; sc-20158), brain natriuretic peptide (BNP; sc-271185), S1PR2 (sc-25491) and GAPDH (sc-32233) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activity Assay, Western Blot, Expressing, Control, Negative Control, Small Interfering RNA

Journal: International journal of molecular medicine

Article Title: Sphingosine-1-phosphate ameliorates the cardiac hypertrophic response through inhibiting the activity of histone deacetylase-2.

doi: 10.3892/ijmm.2017.3325

Figure Lengend Snippet: Figure 8. Model of the mechanisms underlying the ameliorative effects of S1P on cardiac hypertrophy. S1P treatment inhibited HDAC2 activity, resulting in increased histone acetylation and upregulation of KLF4, thus ameliorating cardiac hypertrophy. Cardiac hypertrophy is potentially also mediated by S1PR2. These effects may contribute to the improvement of cardiac function. HDAC2, histone deacetylase‑2; KLF4, Krüppel‑like factor 4; S1P, sphingosine‑1‑phos- phate; S1PR2, S1P receptor 2.

Article Snippet: Antibodies against atrial natriuretic peptide (ANP; sc-20158), brain natriuretic peptide (BNP; sc-271185), S1PR2 (sc-25491) and GAPDH (sc-32233) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activity Assay

Journal: Kidney international

Article Title: Ubiquitin-dependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury.

doi: 10.1038/sj.ki.5000261

Figure Lengend Snippet: Figure 5 | Immunoblot analysis and effects of Smurf2 immunodepletion on ubiquitination activity against SnoN and Ski. (a) Western blot analysis demonstrated that Smurf2 was weakly expressed in sham-operated kidneys. In UUO, the increases of Smurf2 were noted in almost inverse proportion to the levels of SnoN. (b) Renal extracts from obstructed or sham-operated kidneys (input) were pre-incubated with anti-Smurf2 antibody and protein G–Sepharose. After centrifugation, the remnant amounts of Smurf2 in the supernatants were checked by immunoblotting using anti-Smurf2 antibody (aSmurf2), and then subjected to the following assay. As a control, we used renal extracts that had been pre-incubated with rabbit immunoglobulin G (aIgG). (c, left panel) In obstructed kidneys, significant smeared bands were observed when HA-tagged SnoN was incubated with the control extracts that had not been immunodepleted of Smurf2 (; lanes 3 and 4); however, the bands were much weaker when HA-tagged SnoN was incubated with Smurf2-immunodepleted extracts ( þ ; lanes 5 and 6). In sham-operated kidneys, no significant bands were detected (lanes 1 and 2). (Right panel) We also performed a similar ubiquitination assay for Ski. No notable differences in the intensity of the bands were observed between the control (; lanes 9 and 10) and the Smurf2-immunodepleted extracts ( þ ; lanes 11 and 12) in obstructed kidneys.

Article Snippet: Equal amounts of proteins (40 mg) were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously.29–31 The primary antibodies were goat anti-human SnoN (Santa Cruz Biotechnology), rabbit anti-human c-Ski (Santa Cruz Biotechnology), rabbit antihuman Smurf2 (sc-25511; Santa Cruz Biotechnology), and mouse monoclonal anti-b-actin (Sigma, St Louis, MO, USA). b-Actin was used as an internal control.

Techniques: Western Blot, Immunodepletion, Ubiquitin Proteomics, Activity Assay, Incubation, Centrifugation, Control

Journal: Oncogene

Article Title: 15-Lipoxygenase-2 gene regulation by its product 15-(S)-hydroxyeicosatetraenoic acid through a negative feedback mechanism that involves peroxisome proliferator-activated receptor gamma.

doi: 10.1038/sj.onc.1209617

Figure Lengend Snippet: Figure 5 Electrophoretic mobility shift analysis of peroxisome proliferator-activated receptor g (PPARg) and RORa1 protein binding to the 32P-labeled-15 lipoxygenase-2 (15-LOX-2) oligonu- cleotide (560 to 596 bp) in Prostate epithelial cells (PrEC) and Prostate carcinoma cells (PC-3). The protein-DNA complexes that were supershifted (arrows) by the addition of an antibody that recognizes PPARg. (a) Lanes 1 and 2, PrEC nuclear extract without and with dnPPARg; lanes 3 and 4, PC-3 nuclear extract without and with dnPPARg; lane 5, PC-3 nuclear extract with the consensus PPARg probe; lane 6, PC-3 nuclear extract incubated with the mutant PPARg probe. Consensus PPRE or mutant oligonucleotides were used as unlabeled competitors to determine the specificity of the binding reaction in PC-3. (b) Lane 1, 32P- labeled-RORE consensus site free probe; lanes 2, 3 and 4, PC-3 nuclear extract with increasing concentrations of unlabelled RORE oligonucleotide as competitor; lane 5, 32P-labeled-15-LOX-2 free probe; lanes, 6, 7 and 8, PC-3 nuclear extract in the presence of increasing concentrations of unlabeled 15-LOX-2 oligonucleotide as competitor; lanes, 9 and 10, PC-3 nuclear extract with 15-LOX-2 probe and without and with the RORa antibody; lane 11, PC-3 nuclear extract with 15-LOX-2 probe and RORa antibody in presence of 50 unlabeled 15-LOX-2 oligonucleotide.

Article Snippet: Wild-type PPARg and mutant gel shift oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Electrophoretic Mobility Shift Assay, Protein Binding, Labeling, Incubation, Mutagenesis, Binding Assay