Journal: bioRxiv

Article Title: Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

doi: 10.64898/2026.03.13.711548

Figure Lengend Snippet: (A) Representative immunofluorescence images of primary cilia in healthy control (HC), systemic sclerosis (SSc), and VEDOSS (very early SSc) dermal fibroblasts under basal conditions (CTR) or following 24 h TGFβ stimulation. Acetylated α-tubulin marks the axoneme (red) and nuclei are stained with DAPI (blue). (B) Quantification of primary cilium length in HC, SSc, and VEDOSS fibroblasts under basal conditions. n=3 donors per group, a minimum of 100 cilia were quantified per sample. (C) Time course of TGFβ-induced cilia shortening in HC and SSc fibroblasts. n=3 donors per group, a minimum of 100 cilia were quantified per sample. (D) Quantification of cilium length in HC and SSc fibroblasts subjected to a 24 h TGFβ pulse followed by washout and recovery in 0.5% serum for 24 and 48 h. n=3 experimental repeats. (E) Quantification of cilium length in HC and SSc fibroblasts after sequential serum starvation, TGFβ exposure, and two passages in 10% serum. n=3 experimental repeats. (F) Cilium length in HC and SSc fibroblasts treated with the TGFβ receptor inhibitor SD208 in the presence or absence of exogenous TGFβ. n=3 experimental repeats. (G) Representative western blot of pSMAD3, total SMAD3, and βactin in HC and SSc fibroblasts treated with TGFβ and/or SD208. n=3 donors per group. (H) Quantification of cilium length in HC and SSc fibroblasts cultured in full growth medium in the presence of DMSO (CTR) or SD208 for 2 passages. n=3 experimental repeats. All data panels were analysed by ANOVA (ns, non-significant; ** P<0.01; *** P<0.001; ****P<0.0001).

Article Snippet: Proteins were transferred onto Hybond nitrocellulose membranes (Amersham) and probed with antibodies specific for αSMA (Abcam ab7817), β-Actin (Sigma A5441), CAV1 (Santa Cruz sc894), p53 (Santa Cruz sc126), acetylated-α-Tubulin (Cell Signaling 5335), MLC2 (Cell Signaling 8505), pMLC2 (Ser19) (Cell Signaling 3674), ppMLC2 (Ser19, Thr18) (Cell Signaling 3674), SMAD3 (Cell Signalling 9523), pSMAD3 (Abcam ab52903).

Techniques: Immunofluorescence, Control, Staining, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

doi: 10.64898/2026.03.13.711548

Figure Lengend Snippet: (A) Top: representative western blot showing the effect of SMAD3 siRNA (siSMAD3) on total SMAD3 protein level and on TGFβ-induced SMAD3 phosphorylation in healthy control (HC) and systemic sclerosis (SSc) fibroblasts, compared to scrambled siRNA (siSCR). Bottom: Densitometric quantification of SMAD3 in control (siSCR) and siSMAD3-treated HC and SSc fibroblasts (n=3 individual donors per group). (B) Quantification of cilium length in HC and SSc fibroblasts treated with siSCR or siSMAD3 and stimulated for 24 h with TGFβ. n=3 individual donors per group. (C) Top: representative western blots showing pMLC2, ppMLC2, and total MLC2, in response to treatments TGFβ with or without KD025 in three HC donor and three SSc donor fibroblast lines. Bottom: Densitometric quantification of pMLC2 normalised to MLC2 in the same condition. (D) Cilium length in HC and SSc fibroblasts treated with the ROCK2 inhibitor KD205 ± TGFβ. Mean cilia length was measured in three HC donor and three SSc donor fibroblast lines treated as indicated. All data panels were analysed by ANOVA (ns, non-significant; * P<0.05; ****P<0.0001).

Article Snippet: Proteins were transferred onto Hybond nitrocellulose membranes (Amersham) and probed with antibodies specific for αSMA (Abcam ab7817), β-Actin (Sigma A5441), CAV1 (Santa Cruz sc894), p53 (Santa Cruz sc126), acetylated-α-Tubulin (Cell Signaling 5335), MLC2 (Cell Signaling 8505), pMLC2 (Ser19) (Cell Signaling 3674), ppMLC2 (Ser19, Thr18) (Cell Signaling 3674), SMAD3 (Cell Signalling 9523), pSMAD3 (Abcam ab52903).

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Smad2 Protects against TGF-?/Smad3-Mediated Renal Fibrosis

doi: 10.1681/ASN.2009121244

Figure Lengend Snippet: Deletion of Smad2 enhances Smad3 phosphorylation in the UUO kidney and in vitro in response to TGF-β1. (A and B) Immunohistochemistry and Western blot analysis show that compared with the UUO kidney of Smad2ff mice, numbers of nucleated phospho-Smad3–positive cells and phospho-Smad3 protein are markedly increased in the UUO kidney of conditional Smad2 KO mice. (C) Knockdown of Smad2 from TECs (NRK52E) results in a significant increase in phospho-Smad3 at 30 minutes (peak time) after TGF-β1 stimulation. (D) Western blot analysis shows that compared with Smad2 WT MEFs, addition of TGF-β1 (2 ng/ml) largely enhances Smad3 phosphorylation in Smad2 KO MEFs in a time-dependent manner when compared with the Smad2 WT MEFs. Data are means ± SEM for groups of eight mice in vivo and four independent experiments in vitro. *P < 0.05, **P < 0.01, ***P < 0.001 versus time (dosage) 0 or normal; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Smad2WT MEFs or injured Smad2ff (UUO) mice.

Article Snippet: 36 The antibodies used in this study included phospho-Smad3 (Rockland Immunochemicals, Gilbertsville, PA), CTGF, TGF-β1 (Santa Cruz Biotechnology), and collagen I and collagen III (Southern Tech, Birmingham, AL).

Techniques: In Vitro, Immunohistochemistry, Western Blot, In Vivo

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Smad2 Protects against TGF-?/Smad3-Mediated Renal Fibrosis

doi: 10.1681/ASN.2009121244

Figure Lengend Snippet: Deletion of Smad2 enhances Smad3 signaling in MEFs. (A) Immunofluorescence detects that MEFs lacking Smad2 substantially enhance phosphorylated Smad3 nuclear translocation at 30 minutes after TGF-β1 (2 ng/ml) stimulation when compared with the Smad2 WT MEFs. (B) Quantitative analysis of phospho-Smad3 nuclear translocation in response to TGF-β1 (2 ng/ml). (C) Smad3 promoter activity assay. (D) ChIP assay for binding of Smad3 to the COL1A2 promoter. (E) Quantitative real-time PCR analysis of the binding of Smad3 to COL1A2. Data are means ± SEM for four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus time 0 or empty vector control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Smad2 WT MEFs.

Article Snippet: 36 The antibodies used in this study included phospho-Smad3 (Rockland Immunochemicals, Gilbertsville, PA), CTGF, TGF-β1 (Santa Cruz Biotechnology), and collagen I and collagen III (Southern Tech, Birmingham, AL).

Techniques: Immunofluorescence, Translocation Assay, Activity Assay, Binding Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Smad2 Protects against TGF-?/Smad3-Mediated Renal Fibrosis

doi: 10.1681/ASN.2009121244

Figure Lengend Snippet: Overexpression of Smad2 attenuates the fibrotic effect of TGF-β1 in TECs. (A) Characterization of Smad2-overexpressing TECs. (B) Western blot analysis shows that compared with empty vector control (VC), overexpression of Smad2 (S2Over) inhibits TGF-β1–induced (2 ng/ml) phosphorylation of Smad3 in TECs (NRK52E). (C) Real-time PCR shows that overexpression of Smad2 in TECs attenuates the fibrosis response to TGF-β1 (2 ng/ml, 3 hours), including a significant inhibition of TGF-β1, CTGF, collagens I and III, and TIMP mRNA expression, while increasing MMP-2 expression. Data are means ± SEM for four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus time 0 or empty vector control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus empty vector control (VC).

Article Snippet: 36 The antibodies used in this study included phospho-Smad3 (Rockland Immunochemicals, Gilbertsville, PA), CTGF, TGF-β1 (Santa Cruz Biotechnology), and collagen I and collagen III (Southern Tech, Birmingham, AL).

Techniques: Over Expression, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Inhibition, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Diterpenoid alkaloids isolated from Delphinium trichophorum alleviate pulmonary fibrosis via the TGF-β/Smad pathway in 3T6 and HFL-1 cells.

doi: 10.1016/j.biopha.2022.112906

Figure Lengend Snippet: Fig. 7. The effects of DTF1 and DTF2 on regulating the TGF-β/Smad pathway. (A) Representative Western blotting images of TGF-β1, α-SMA, Smad3, and p-Smad3. The protein expression of (B) α-SMA, (C) TGF-β1, (E) Smad3, and (F) p-Smad3/Smad3 were determined. (D) Representative Western blotting images of Smad4, p- Smad4, and Smad7. The protein expression of (G) Smad4, (H) p-Smad4/Smad4, and (I) Smad7 were determined. ##P < 0.01 compared with control. #P < 0.05 compared with the control. **P < 0.01 compared with the model. *P < 0.05 compared with the model. TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin.

Article Snippet: Reagents were procured from several suppliers, these included lipopolysaccharide (LPS) from Sigma-Aldrich (St. Louis, MO, USA); recombinant transforming growth factor-β1 (TGF-β1) from PeproTech (Rocky Hill, NJ, USA); primary antibodies against TGF-β1, α-SMA, Smad3, Smad4, p-Smad3, p-Smad4, Smad7, and GAPDH from Abcam (Cambridge, UK); and the enhanced chemiluminescence (ECL) reagent from Bio-Rad (Hercules, CA, USA).

Techniques: Western Blot, Expressing, Control

Journal: Molecular medicine reports

Article Title: Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

doi: 10.3892/mmr.2014.2912

Figure Lengend Snippet: Figure 1. Analysis of Smad3/4 complex binding to the β3GalT4 promoter. (A) Smad4 binding site (5'‑GTCTAGAC‑3') located between positions ‑788 and ‑795, relative to the transcriptional start point of the β3GalT4 promoter. (B) EMSA. Each lane contained 0.3 nM labeled probe. Smad4 protein (900 ng) was loaded. Smad3 (400, 600, 800 and 1000 ng) was loaded on lanes 3‑6, respectively. (B‑D) Black arrow indicates retarded DNA fragments; white arrow indicates free probes. (C) EMSA to eliminate the nonspecific binding of the Smad3/4 proteins. The labeled probe and 100 or 200‑fold excess of the unlabeled probe were used in competitive assays. A fragment of actin promoter was used as a nonspecific probe. (D) Determination of the binding sites of Smad3/4. Top: Nucleotide sequence of part of the β3GalT4 promoter region and SBE. All probes used were 40 bp. NotI sites were generated at the SBE site to produce a mutated probe. Underlined nucleotides were changed: Bottom: EMSA using the unlabeled probe and mutated or intact DNA fragment. (E) ChIP assays. Input DNA, a DNA fragment immunoprecipitated with anti‑Smad4 antibody (lane ‘S’) and a DNA fragment immunoprecipitated with IgG (lanes ‘‑’) were used as templates for the polymerase chain reaction. A nonspecific DNA region was used as a negative control. Anti‑Pol II antibodies that immunoprecipitated the Pol II‑actin promoter complexes were used as a positive control. β3GalT4, β1,3-galactosyltransferase‑4; EMSA, electrophoretic mobility shift assay; SBE, Smad4 binding element.

Article Snippet: The primary antibodies used were mouse anti-E-cad IgG2a monoclonal antibody and mouse-anti-β catenin IgG1 monoclonal antibody (BD Biosciences, San Jose, CA, USA), mouse anti-N-cad IgG1 and rabbit anti-RNA polymerase II (Pol II) IgG polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-vimentin IgG1 monoclonal antibody and anti-β tubulin IgG1 monoclonal antibody (Sigma-Aldrich), rabbit anti-Smad3 mAb IgG monoclonal antibody and anti-Smad4 polyclonal antibody (Cell Signaling Technology, Boston, MA, USA).

Techniques: Binding Assay, Labeling, Sequencing, Generated, Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Positive Control, Electrophoretic Mobility Shift Assay

Journal: Molecular medicine reports

Article Title: Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

doi: 10.3892/mmr.2014.2912

Figure Lengend Snippet: Figure 2. Effect of Smad3 or Smad4 overexpression in NMuMG cells. (A) Morphological changes. Cells were cultured in 6‑well plates and treated with 2 ng/ ml TGFβ for 48 h. Images were captured under phase‑contrast microscopy. (B and C) Expression of EMT markers in the transfected cells. Cells were cultured in 6‑well plates and treated with or without 2 ng/ml TGFβ for 48 h. The cells were harvested and lysed in radioimmunoprecipitation assay buffer. Lysates (10 µg protein/well) were subjected to SDS‑PAGE and western blot analysis and the expression of the markers N‑cad, vimentin, E‑cad and β‑catenin was analyzed by western blot analysis, as described in Materials and methods. Representative western blot analysis results and expression levels relative to Tubulin are expressed as the mean ± standard deviation from triplicate experiments. *0.01

Article Snippet: The primary antibodies used were mouse anti-E-cad IgG2a monoclonal antibody and mouse-anti-β catenin IgG1 monoclonal antibody (BD Biosciences, San Jose, CA, USA), mouse anti-N-cad IgG1 and rabbit anti-RNA polymerase II (Pol II) IgG polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-vimentin IgG1 monoclonal antibody and anti-β tubulin IgG1 monoclonal antibody (Sigma-Aldrich), rabbit anti-Smad3 mAb IgG monoclonal antibody and anti-Smad4 polyclonal antibody (Cell Signaling Technology, Boston, MA, USA).

Techniques: Over Expression, Cell Culture, Microscopy, Expressing, Transfection, Radio Immunoprecipitation, Western Blot, Standard Deviation

Journal: Frontiers in endocrinology

Article Title: Investigating the role of ASCC1 in the causation of bone fragility.

doi: 10.3389/fendo.2023.1137573

Figure Lengend Snippet: FIGURE 7 Mutant ASCC1 directs MSC fate toward adipocytes by increasing adipogenic markers PPARG and FASN and away from osteoblasts by decreasing osteogenic markers RUNX2, ALPL, and CTNNB1. Mutant ASCC1 inhibits TGF-b/SMAD signaling when stimulated with recombinant human TGF-b1, through downregulating the expression ratio of pSMAD3/SMAD3 in patient fibroblasts. Figure created with BioRender.com.

Article Snippet: After incubating the membranes with a blocking reagent (Bio-Rad, Vienna, Austria), they were probed with the following primary antibodies diluted in blocking buffer overnight: monoclonal anti-rabbit COL1A1 (1:1,000, Cell Signaling, Leiden, NL), polyclonal anti-rabbit COL1A2 (1:1,000, Abcam, Amsterdam, NL), monoclonal anti-rabbit actin (1:2,000, Cell Signaling, Leiden, NL), monoclonal anti-rabbit pSmad3 (Ser423/425, 1:1,000, Cell Signaling, Leiden, NL), and monoclonal anti-rabbit Smad3 (1:1,000; Cell Signaling, Leiden, NL).

Techniques: Mutagenesis, Recombinant, Expressing

Journal: Molecular Medicine Reports

Article Title: Molecular responses of radiation-induced liver damage in rats

doi: 10.3892/mmr.2014.3051

Figure Lengend Snippet: Immunohistochemical staining of liver sections 3 days and 1, 2, 4, 8 and 12 weeks after 20 Gy irradiation. Brown color denotes positivity. The perivenous area was the major area of positive staining. TGF-β1, Smad3, CTGF and NF-κB p65 staining was positive between 3 days and 12 weeks after irradiation. Smad4 and Smad7 staining was positive between 3 days and 1 week after irradiation. TNF-α staining was positive between 1 and 4 weeks after irradiation. (Magnification, ×200). Gy, grays; TGF-β1, transforming growth factor-β1; CTGF, connective tissue growth factor; TNFα, tumor necrosis factor-α, NF, nuclear factor.

Article Snippet: The primary antibodies used were as follows: Rabbit monoclonal antibodies against transforming growth factor-β1 (TGF-β1; 1:250), nuclear factor (NF)-κB65 (1:100), mothers against decapentaplegic homolog 4 (Smad4) (1:40), Smad3 (1:250), Smad7 (1:30), tumor necrosis factor-α (TNF-α; 1:200) and connective tissue growth factor (CTGF; 1:200), all purchased from Boster Co., Ltd.

Techniques: Immunohistochemical staining, Staining, Irradiation

Journal: Molecular Medicine Reports

Article Title: Molecular responses of radiation-induced liver damage in rats

doi: 10.3892/mmr.2014.3051

Figure Lengend Snippet: Western blot analysis (ratio of the molecules investigated, vs. GAPDH). Similar to the results obtained using the reverse transcription quantitative polymerase chain reaction, the protein expression levels were as follows: NF-κB p65 was increased between 3 days and 12 weeks after irradiation. CTGF and Smad3 were significantly increased between 2 and 12 weeks after irradiation. TGF-β1 was significantly increased between 1 and 12 weeks after irradiation and TNF-α was significantly increased between 3 days and 4 weeks after irradiation. Smad4 was significantly increased between 3 days and 1 week after irradiation. Smad7 was significantly increased 3 days after irradiation and reduced significantly 1 and 2 weeks after irradiation, however, it remained higher than that in the control. From 4 weeks post-irradiation, the protein expression of Smad7 returned to the control level. * P<0.05, ** P<0.001 compared with the control group (0 Gy). Gy, grays; CTGF, connective tissue growth factor; TGF-β1, transforming growth factor-β1; TNFα, tumor necrosis factor-α; Smad, mothers against decapentaplegic; NF, nuclear factor.

Article Snippet: The primary antibodies used were as follows: Rabbit monoclonal antibodies against transforming growth factor-β1 (TGF-β1; 1:250), nuclear factor (NF)-κB65 (1:100), mothers against decapentaplegic homolog 4 (Smad4) (1:40), Smad3 (1:250), Smad7 (1:30), tumor necrosis factor-α (TNF-α; 1:200) and connective tissue growth factor (CTGF; 1:200), all purchased from Boster Co., Ltd.

Techniques: Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Irradiation, Control

Journal: Molecular Medicine Reports

Article Title: Molecular responses of radiation-induced liver damage in rats

doi: 10.3892/mmr.2014.3051

Figure Lengend Snippet: mRNA expression levels were determined using reverse transcription quantitative polymerase chain reaction (ratio of the molecules investigated, vs. GAPDH). The mRNA expression of NF-κB p65 was upregulated between 3 days and 12 weeks after irradiation. The mRNA expression levels were as follows: CTGF and Smad3 were significantly upregulated between 2 and 12 weeks after irradiation. TGF-β1 was significantly upregulated between 1 and 12 weeks after irradiation. TNF-α was significantly upregulated between 3 days and 4 weeks after irradiation. Smad4 was significantly upregulated 3 days and 1 week after irradiation. Smad7 was significantly upregulated 3 days after irradiation and reduced significantly 1–2 weeks after irradiation, however, the expression levels remained higher than that in the control. From 4 weeks post-irradiation, the mRNA expression of Smad7 returned to the control level. * P<0.05, ** P<0.001, compared with the control group (0 Gy). CTGF, connective tissue growth factor; TGF-β1, transforming growth factor-β1; TNFα, tumor necrosis factor-α, Smad, mothers against decapentaplegic; NF, nuclear factor.

Article Snippet: The primary antibodies used were as follows: Rabbit monoclonal antibodies against transforming growth factor-β1 (TGF-β1; 1:250), nuclear factor (NF)-κB65 (1:100), mothers against decapentaplegic homolog 4 (Smad4) (1:40), Smad3 (1:250), Smad7 (1:30), tumor necrosis factor-α (TNF-α; 1:200) and connective tissue growth factor (CTGF; 1:200), all purchased from Boster Co., Ltd.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Irradiation, Control

Journal: Scientific Reports

Article Title: Elevated serum 1,25(OH) 2 -vitamin D 3 level attenuates renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction in kl/kl mice

doi: 10.1038/srep06563

Figure Lengend Snippet: (a) Immunofluorescence for phosphorylated (p)-Smad3. Scale bar: 100 μm (b) Comparison of the number of pSmad3-positive nuclei among WT, HT, and kl/kl kidneys. (c) Western blot for pSmad3 in UUO-treated kidneys. Full-length blots are included in the . (d) Comparison by quantification of pSmad3 in western blot among WT, HT, and kl/kl kidneys. (e) The amount of TGF-β1 protein in WT, HT, and kl/kl kidneys as measured by ELISA. (f) TGF-β1 mRNA level as measured by real-time PCR. Note that UUO-induced TGF-β signaling was enhanced in HT kidneys compared to WT kidneys and was ameliorated in kl/kl kidneys. Data are presented as the means ± SD. *P < 0.05 (n = 5).

Article Snippet: The primary antibodies were rabbit anti-pSmad3 (Acris Antibodies, Herford, Germany), rat anti-E-cadherin (Sigma Aldrich), mouse anti-αSMA (Sigma Aldrich), rabbit anti-Smad3 (Abcam), mouse anti-β-actin (Sigma Aldrich), and goat anti-GAPDH(Santa Cruz).

Techniques: Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Elevated serum 1,25(OH) 2 -vitamin D 3 level attenuates renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction in kl/kl mice

doi: 10.1038/srep06563

Figure Lengend Snippet: (a) Representative western blots for pSmad3, Smad3, E-cadherin (E-cad), αSMA, and β-actin. Full-length blots are included in the . (b–d) Comparison by quantification of western blots for pSmad3 (b), E-cadherin (c), and αSMA (d). (e–g) Real-time PCR for E-cadherin (e), Col1a1 (f), and αSMA (g). Note that unlike in vivo, no difference in EMT markers was detected between HT and kl/kl cultured proximal tubular cells. Data are presented as the means ± SD. *P < 0.05 compared to the HT group at the same time point. Experiments were repeated in triplicate.

Article Snippet: The primary antibodies were rabbit anti-pSmad3 (Acris Antibodies, Herford, Germany), rat anti-E-cadherin (Sigma Aldrich), mouse anti-αSMA (Sigma Aldrich), rabbit anti-Smad3 (Abcam), mouse anti-β-actin (Sigma Aldrich), and goat anti-GAPDH(Santa Cruz).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, In Vivo, Cell Culture

Journal: Scientific Reports

Article Title: Elevated serum 1,25(OH) 2 -vitamin D 3 level attenuates renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction in kl/kl mice

doi: 10.1038/srep06563

Figure Lengend Snippet: (a) Representative western blots for pSmad3, Smad3, E-cadherin (E-cad), αSMA, and β-actin. Full-length blots are included in the . (b–d) Comparison by quantification of western blots for pSmad3 (b), E-cadherin (c) and αSMA (d). (e-g) Real-time PCR for E-cadherin (e), Col1a1 (f), and αSMA (g). A high concentration of phosphate does not enhance TGF-β1-induced EMT. Note that basal Smad3 phosphorylation and αSMA protein expression are enhanced in the presence of a high phosphate level. Data are presented as the means ± SD. *P < 0.05. Experiments were repeated in triplicate.

Article Snippet: The primary antibodies were rabbit anti-pSmad3 (Acris Antibodies, Herford, Germany), rat anti-E-cadherin (Sigma Aldrich), mouse anti-αSMA (Sigma Aldrich), rabbit anti-Smad3 (Abcam), mouse anti-β-actin (Sigma Aldrich), and goat anti-GAPDH(Santa Cruz).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay, Expressing

Journal: Scientific Reports

Article Title: Elevated serum 1,25(OH) 2 -vitamin D 3 level attenuates renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction in kl/kl mice

doi: 10.1038/srep06563

Figure Lengend Snippet: (a) Representative western blots for pSmad3, Smad3, E-cadherin, αSMA, and β-actin. Full-length blots are included in the . (b–d) Comparison by quantification of western blots for pSmad3 (b), E-cadherin (c), and αSMA (d). (e–j) Real-time PCR results for E-cadherin (e, h), Col1a1 (f, i), and αSMA (g, j) in the presence of either FGF23 (e, f, g) or calcitriol (h, i, j). FGF23 have no effect on TGF-β1-induced EMT at neither low nor high concentration. Note that calcitriol ameliorates TGF-β1 induced EMT in a dose-dependent manner. Data are presented as the means ± SD. *P < 0.05 versus cells treated with TGF-β1 only. Experiments were repeated in triplicate.

Article Snippet: The primary antibodies were rabbit anti-pSmad3 (Acris Antibodies, Herford, Germany), rat anti-E-cadherin (Sigma Aldrich), mouse anti-αSMA (Sigma Aldrich), rabbit anti-Smad3 (Abcam), mouse anti-β-actin (Sigma Aldrich), and goat anti-GAPDH(Santa Cruz).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: Scientific Reports

Article Title: Elevated serum 1,25(OH) 2 -vitamin D 3 level attenuates renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction in kl/kl mice

doi: 10.1038/srep06563

Figure Lengend Snippet: (a) Representative immunofluorescence for phosphorylated (p)-Smad3. (b) Western blot for pSmad3 in UUO- or sham-treated kidneys. Full-length blots are included in the . (c) Comparison of the number of pSmad3-positive nuclei. (d) Comparison of pSmad3 to total Smad3 ratio in western blots. (e) TGF-β1 protein expression in the kidneys as measured by ELISA. (f) TGF-β1 mRNA expression as measured by real-time PCR. Amelioration of UUO-induced activation of TGF-β1 signaling in kl/kl mice was abolished by a vitamin D3 free diet. pSmad3 positive nuclei and TGF-β1 expression were increased at the protein and mRNA levels. Data are presented as the means ± SD. *P < 0.05 (n = 5), #p < 0.05 vs. sham treated kidneys. &P < 0.05 vs. D-diet kl/kl mice.

Article Snippet: The primary antibodies were rabbit anti-pSmad3 (Acris Antibodies, Herford, Germany), rat anti-E-cadherin (Sigma Aldrich), mouse anti-αSMA (Sigma Aldrich), rabbit anti-Smad3 (Abcam), mouse anti-β-actin (Sigma Aldrich), and goat anti-GAPDH(Santa Cruz).

Techniques: Immunofluorescence, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activation Assay