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: Oncogene

Article Title: Low dose IR-induced IGF-1-sCLU expression: a p53-repressed expression cascade that interferes with TGFβ1 signaling to confer a pro-survival bystander effect

doi: 10.1038/onc.2012.64

Figure Lengend Snippet: In A, B, Ten week-old (18 gm) female FVB/N mice were exposed to IR (0.1 or 1.0 Gy) and colon and bone marrow tissues extracted at various times as indicated. In C, D, Dose-response (0.1 – 1.0 Gy) studies were also performed in female FVBN hCLUp-Luc transgenic mice and reporter expression monitored at specific times (24 or 72 h) as indicated. In A–D, Data (means +SE, n=6) are from experiments performed at least two independent times with three mice/group each. Tissues were pooled and then analyzed. In E, F, Steady state protein level changes in psCLU and sCLU, phosphorylated or total Smad3 (P-Smad3 or t-Smad3, respectively), γ-H 2 AX and α-tubulin were monitored by Western analyses. Note increased levels of P-Smad3/tSmad3, indicative of activated TGFβ1 signaling after 0.1 or 1.0 Gy at 24 or 72 h post-IR in both colon and bone marrow tissue samples. Activated Smad signaling (indicated by elevated P-Smad3/t-Smad3 levels) corresponded well with increased sCLU protein levels. Shown are representative Western blots for colon and bone marrow from experiments performed three times in triplicate with similar results.

Article Snippet: Antibodies to phosphorylated Smad3 (p-Smad3, C25A9) or total Smad3 (t-Smad3, C67H9) were from Cell Signaling (Billerica, MA).

Techniques: Transgenic Assay, Expressing, Western Blot

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