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: bioRxiv

Article Title: Human-specific NOTCH -like genes in a region linked to neurodevelopmental disorders affect cortical neurogenesis

doi: 10.1101/221226

Figure Lengend Snippet: (A) Location of NOTCH2NL genes in chromosome 1q21.1 flanking the 1q21.1 distal deletion/duplication syndrome locus. This region contains many additional genes derived from human segmental duplication (shaded light blue). TAR syn is Thrombocytopenia Absent Radius syndrome. (B) Gene and protein features of NOTCH2 and NOTCH2NL. (C) Results of the de novo assembly of NOTCH2NL loci for H9 human embryonic stem cells and relative allele expression from week 5 cortical organoids as measured by full-length cDNA sequencing. * Not enough nucleotide differences present to distinguish between the two NOTCH2NL sh2ntdel alleles, so counts evenly split between the two. (D) Observed NOTCH2NL paratypes (n=72, counting paratypes transmitted from parent to child only once) obtained from linked-read sequencing and assembly of 15 individuals. See also Fig. S1 and Table S1 and S4.

Article Snippet: 2 pg of one specific antibody was added (anti-HA Abcam ab9110, anti- Myc Abcam ab9E10, anti-His Abcam ab9108, anti-NOTCH2 SCBT sc25-255) and incubated overnight at 4°C in a rotating wheel.

Techniques: Derivative Assay, Expressing, Sequencing

Journal: bioRxiv

Article Title: Human-specific NOTCH -like genes in a region linked to neurodevelopmental disorders affect cortical neurogenesis

doi: 10.1101/221226

Figure Lengend Snippet: (A) Coverage of Illumina genome sequencing reads mapped to the NOTCH2 locus demonstrating an excess of coverage in the 5’ region of NOTCH2 into intron 4 in all hominins examined, chimpanzee and gorilla, but not orangutan. (B) Schematic of NOTCH2NL-containing genes identified in gorilla, chimpanzee and human demonstrating that only in human NOTCH2NL genes encode NOTCH2-related proteins. (C) Immunoblot using an N-terminal NOTCH2 antibody (aa 25-255) of protein extracts from human and chimpanzee pluripotent stem cells showing expression of full-length NOTCH2 in both samples, but NOTCH2NL only in human. (D) Summary of the major events leading to the emergence of NOTCH2NL-related genes in the great ape lineage. See also Figure S2.

Article Snippet: 2 pg of one specific antibody was added (anti-HA Abcam ab9110, anti- Myc Abcam ab9E10, anti-His Abcam ab9108, anti-NOTCH2 SCBT sc25-255) and incubated overnight at 4°C in a rotating wheel.

Techniques: Sequencing, Western Blot, Expressing

Journal: bioRxiv

Article Title: Human-specific NOTCH -like genes in a region linked to neurodevelopmental disorders affect cortical neurogenesis

doi: 10.1101/221226

Figure Lengend Snippet: Radial glia-specific expression of NOTCH2NL in human fetal brain samples. Scatterplot of 3466 fetal brain cells after principal components analysis and t-stochastic neighbor embedding (tSNE) samples as described in Nowakowski, et al, 2017. Cells are colored by annotated cell type clusters (A), NOTCH2NL expression (B) and NOTCH2 expression (C). See also Figure S3.

Article Snippet: 2 pg of one specific antibody was added (anti-HA Abcam ab9110, anti- Myc Abcam ab9E10, anti-His Abcam ab9108, anti-NOTCH2 SCBT sc25-255) and incubated overnight at 4°C in a rotating wheel.

Techniques: Expressing

Journal: bioRxiv

Article Title: Human-specific NOTCH -like genes in a region linked to neurodevelopmental disorders affect cortical neurogenesis

doi: 10.1101/221226

Figure Lengend Snippet: (A)Schematic showing NOTCH2NL alleles present in control (H9*) and NOTCH2NL mutant (H9 NOTCH2NLΔ ) cell lines. (B). Multi-region UCSC genome browser view of NOTCH2 and NOTCH2NL genes with tracks showing normalized genome sequencing coverage demonstrating homozygous loss of exon 2-5 sequence for NOTCH2NLA and NOTCH2NLB and heterozygous loss for NOTCH2NLC . Some coverage is seen in NOTCH2NLA and NOTCH2NLB due to a small portion of ambiguous linked read barcodes. Schematics of the cortical organoid protocol used (C) with pictures showing cells at various stages (D) and cell types generated. (F-I) Immunofluorescence staining of H9 hESC cortical organoids with markers of radial glia (PAX6, BLBP), intermediate progenitor cells (TBR2) and deep layer excitatory projection neurons (TBR1, CTIP2). (j) Spearman’s rank correlation plot of the top 250 upregulated and downregulated genes (H9 NOTCH2NLΔ / H9*), and the matching data in W3, W4 and W5 H9 organoids. Numbers in plot indicate pairwise correlation values. (K) Heatmap showing expression profiles for a selection of genes in the significantly enriched GO cluster ‘neuron differentiation’. n = 3.

Article Snippet: 2 pg of one specific antibody was added (anti-HA Abcam ab9110, anti- Myc Abcam ab9E10, anti-His Abcam ab9108, anti-NOTCH2 SCBT sc25-255) and incubated overnight at 4°C in a rotating wheel.

Techniques: Mutagenesis, Sequencing, Generated, Immunofluorescence, Staining, Expressing, Selection

Journal: bioRxiv

Article Title: Human-specific NOTCH -like genes in a region linked to neurodevelopmental disorders affect cortical neurogenesis

doi: 10.1101/221226

Figure Lengend Snippet: (A) Co-immunoprecipitation of NOTCH2 and NOTCH2NL analyzed by immunoblot. (B-C) Co-transfection of NOTCH2-GAL4 and NOTCH2NL boosts activity of the pGL3-UAS reporter. Upon stimulation of NOTCH2-GAL4 with JAG2 in a coculture setup, NOTCH2NL remains effective in boosting reporter activity. Average of 4 independent experiments with 6 replicates each. Two-way ANOVA with Tukey’s HSD (** p <10 -8 , *** p <10 -12 ), error bars indicate SEM. (D) NOTCH2NL is secreted and can be immunoprecipitated from the medium of mouse ESCs ectopically expressing NOTCH2NL. (E) Addition of NOTCH2NL-conditioned medium to cells transfected with NOTCH2-GAL4 and the pGL3-UAS reporter also enhances reporter activity. Average of 2 independent experiments with 4 and 3 replicates each. One-way ANOVA with Tukey’s HSD (* p < 10 -4 ), error bars indicate SEM. (F-G) The effect of NOTCH2NL is not restricted to NOTCH2, in an assay with NOTCH1-GAL4 we observe similar effects. (H-K) The presence or absence of the ancestral start codon, and the amino acid 197 Thr™»lie variant, each show specific characteristics in the reporter assays. (H-I) Average of 2 independent experiments with 6 replicates each. Two-way ANOVA with Tukey’s HSD (** p < 10 -8 , *** p < 10 -12 ), error bars indicate SEM. (J-K) 6 replicates in one experiment. Student’s t-test with Holm-Bonferroni correction (* p < 0.05, ** p < 10 -3 ), error bars indicate SD. See also Figure S6.

Article Snippet: 2 pg of one specific antibody was added (anti-HA Abcam ab9110, anti- Myc Abcam ab9E10, anti-His Abcam ab9108, anti-NOTCH2 SCBT sc25-255) and incubated overnight at 4°C in a rotating wheel.

Techniques: Immunoprecipitation, Western Blot, Cotransfection, Activity Assay, Expressing, Transfection, Variant 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