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