A canine renal distal tubular epithelium cell line (MDCK) was purchased from the American Type Culture Collection (Rockville, USA). RSG, GW9662, and PHA665752 were obtained from Sigma-Aldrich (St. Louis, MO, USA). The following primary antibodies were used: p-Met, Smad7, TGF-β1, and PPAR-γ (Santa Cruz, USA); HGF (Abcam, USA); p-PPAR-γ (Ser112) (Bioss, China); c-Met (Proteintech, China); Smad2, Smad3, p-Smad2 3, and Lamin B (Wanleibio, China); and GAPDH (Zhongshan Golden Bridge Bio Co., Ltd., China). The secondary antibodies, including HRP-conjugated AffiniPure goat anti-rabbit IgG and HRP-conjugated AffiniPure goat anti-mouse IgG, were purchased from Zhongshan Golden Bridge Bio Co., Ltd. (Beijing, China). A Nuclear and Cytoplasmic Protein Extraction Kit was acquired from Beyotime Institute of Biotechnology (Shanghai, China).
Smad7
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Smad7 is a protein that plays a key role in the transforming growth factor-beta (TGF-β) signaling pathway. It acts as an inhibitory Smad, which negatively regulates the TGF-β signaling cascade.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (10 mentions)
Smad2 3, by Santa Cruz Biotechnology (6 mentions)
Tgf β1, by Santa Cruz Biotechnology (5 mentions)
Smad3, by Santa Cruz Biotechnology (5 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
Smurf2, by Santa Cruz Biotechnology (5 mentions)
E cadherin, by Santa Cruz Biotechnology (5 mentions)
P smad3, by Cell Signaling Technology (4 mentions)
Tgf β, by Santa Cruz Biotechnology (4 mentions)
Phospho smad3, by Cell Signaling Technology (4 mentions)
Smad2, by Cell Signaling Technology (4 mentions)
α sma, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
α sma, by Abcam (3 mentions)
P smad2, by Cell Signaling Technology (3 mentions)
Il 1β, by Abcam (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Smad3, by Cell Signaling Technology (3 mentions)
α sma, by Merck Group (3 mentions)
48 protocols using smad7
1
Exploring Renal Distal Tubule Epithelium Signaling
2
Western Blot Analysis of Cardiac Proteins
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Protein samples were isolated from the left ventricular myocardium of rats. Left ventricular myocardium lysates were prepared by homogenization in cell lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). The protein samples (40/20 µg) were mixed with 2X sodium dodecyl sulfate sample loading buffer (Beyotime Institute of Biotechnology) and were subsequently separated on a 12% polyacrylamide gel and blotted on a nitrocellulose membrane (Beyotime Institute of Biotechnology, Inc). The membranes were blocked with 5% non-fat milk, followed by incubation (at 4°C for 24 h) with antibodies specific for TGF-β1 (1:100, sc-146), TAK1 (1:100, sc-7162), Smad3 (1:100, sc-169248), Smad7 (1:100, sc-100140) or β-actin (1:100; sc-47778) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The membranes were subsequently incubated at 37°C for 30 min with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. ZDR-5306), rabbit anti-goat (cat. no. ZDR-5308) and goat anti-mouse (cat. no. ZDR-5307) immunoglobulin G (1:1,000; all from Zhongshan Goldenbridge Biotechnology Corporation, Beijing, China) and enhanced chemiluminescence detection system (Bio-Rad Laboratories, Hercules, CA, USA) was used for visualization. The grey value was measured using Quantity One software.
3
Western Blot Analysis of BMP2 and Smad7
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Cells were seeded in six-well plates and treated per the experimental design. Total protein was obtained after lysis, and the cleared lysates were denatured by boiling for 10 min with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer. The proteins were separated by electrophoresis with Tris-glycine gels and carefully transferred onto polyvinylidene difluoride (PVDF) membranes in the dark. Then, the PVDF membranes were blocked with 5% evaporated milk for 1 h and incubated overnight with primary antibodies against BMP2 (Abcam, USA) and Smad7 (Santa Cruz, USA). After washing, the membranes were probed with a fluorescently labeled secondary antibodiesy. The immune-reactive signals were detected using a Bio-Rad machine. In addition, the membranes were incubated with a monoclonal mouse anti-human β-actin (Abcam, USA) antibody as a loading control. The relative band intensity was measured using ImageJ analysis software [35 (link), 36 (link)].
4
Western Blot Analysis of Protein Expression
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We lysed total protein from treated cells using RIPA buffer (Cell Signaling Technology) supplemented with Protease Inhibitor Cocktail (Thermo Fisher; 78430). Western blotting was performed as previously described41 (link), and the primary antibodies included GAPDH (Santa Cruz, sc-137179), LKB1 (Santa Cruz, sc-32245), ALKBH5 (Proteintech, 16837-1-AP), CTCF (Santa Cruz, sc-271474), METTL3 (Abcam, ab195352), METTL14 (Abcam, ab220030), FTO (Abcam, ab126605), WTAP (Proteintech, 60188-1-Ig), SOX2 (Cell signaling, #3579), SMAD7 (Santa Cruz, sc-11392), and MYC (Cell signaling, #13987). We performed densitometric analyses of band intensity using ImageQuant TL 8.2 image analysis software (GE Healthcare Life Sciences, USA) and GAPDH was as an internal control.
5
Western Blot Analysis of MyD88 and Smad7
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MyD88 and Smad7 expression were determined by Western blot using antibodies to MyD88 (1:200) or Smad7 (1:300; Santa Cruz Biotechnology) using our previously-described protocol25 (link). Blots were probed with horseradish peroxidase-conjugated secondary antibodies 1:5,000–1:10,000 in blocking buffer for 1 h, and bands were detected using the chemiluminescent method (SuperSignal West Dura, Thermo Scientific, Waltham, MA). Blots were stripped and re-probed with anti-actin (Santa Cruz Biotechnology) to ensure equal loading.
6
Investigating Cellular Signaling Pathways
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NHDFs were treated with UVB irradiation (144 mJ/cm2) and PVE (1, 10, 100 μg/mL) and then checked for alterations in the level of signaling molecules in the MAPK/AP-1, NF-κB, and TGF/Smad pathways, respectively. Cells were lysed using RIPA Lysis buffer (Cell Signaling Technology, Danvers) and the protein concentration was measured using Bradford reagent (Bio-Rad, Hercules, CA) as described by the manufacturer. The proteins were separated on 10% SDS-PAGE and transferred onto PVDF membranes. The membrane was incubated with various primary antibodies after blocking. Protein bands were visualized with enhanced chemiluminescence detection reagents after hybridization with HRP-conjugated secondary antibodies. Antibodies against ERK, phosphor-ERK, JNK, phosphor-JNK, p38, and phosphor-p38, as well as anti-rabbit-HRP and anti-mouse-HRP antibodies were purchased from Cell Signaling Technology, and those against NF-κB p65, c-Fos, phosphor-c-Fos, c-Jun, phosphor-c-Jun, TGF-β1, Smad2/3, phosphor-Smad2/3, Smad7, and β-actin were purchased from Santa Cruz Biotechnology (Dallas). Each experiment was repeated at least three times.
7
Protein Extraction and Western Blot Analysis
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Protein extraction was performed with lysis buffer, radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, China) containing 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, China), and subsequent sonication. After centrifugation, the supernatant was boiled for denaturation and determination of total protein concentration using the BCA protein assay kit (Beyotime Biotechnology, China). Equivalent amounts of protein were loaded for electrophoresis on 7-10% SDS-PAGE gels (Omni-Easy™ One-Step PAGE Gel Fast Preparation Kit, EpiZyme, China) and transferred to polyvinylidene fluoride membranes (PVDF, 0.2 μm, Bio-Rad). After the membranes were blocked with 5% skim milk at room temperature for 1 h, proteins were incubated overnight at 4°C with the following primary antibodies: Sox9 (Zen Bio, 1 : 2000), COL2A1 (Abcam, 1 : 3000), COL10A1 (Santa Cruz Biotechnology; 1 : 1000), Smad7 (Santa Cruz,1 : 1000), and GAPDH (Zen Bio, 1 : 2000). After the membranes were washed with TBST, they were incubated with the corresponding secondary antibodies (goat anti-rabbit IgG, 1 : 10000, Zen Bio) for 1 h at room temperature. Following sequential washing with TBST and TBS, the target proteins were detected by an ECL detection kit (Thermo Fisher Scientific), and ImageJ software was used for quantification of band density.
8
Protein Expression Profiling in Fibrosis
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The proteins level was detected by Western blotting. The primary antibodies were α-SMA, NOX4, NOX2, NLRP3, caspase1, ASC, IL-1β (1:1000; Abcam, Cambridge, MA), p-Smad2, p-Smad3, Smad2/3 (1:1000; Cell Signaling Technology, MA), COL1A, Smad7, Spry1 (1:100; Santa Cruz Biotechnology). β-Actin acted as an internal control.
9
Western Blot Analysis of Smad Proteins
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The lysates were clarified by centrifugation and the supernatants collected. Protein concentrations were determined using the bicinchoninic acid assay (BCA) Protein Assay (Applygen, Beijing, China). Equivalent amounts of tissue protein (80 μg) were resolved on SDS polyacrylamide gels, and transferred by electroblotting to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% (W/V) nonfat milk at room temperature for 1 h, and then incubated overnight at 4°C with the primary antibody against Smad3 (dilution 1: 200, Santa Cruz, CA, USA), Smad7 (dilution 1:200, Santa Cruz, CA, USA), p-Smad3 (dilution 1:1000, Epitomics, CA, USA), and β-actin (dilution 1: 1000, Santa Cruz, CA, USA). After washing in a buffer containing Tris-buffered saline (TBS), with 0.1% Tween, the membranes were incubated with horseradish peroxidase (HRP)-linked anti-mouse secondary antibody at a dilution of 1:3000. Following washing in 0.1% Tween TBS buffer, the immunolabeled proteins were detected by enhanced chemiluminescense reagents (Applygen, Beijing, China). The density of the detected bands was analyzed by Quantity One software.
10
Western Blot Analysis of Fibrosis Markers
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Total proteins were extracted, using RIPA buffer and a protease inhibitor, and quantified using the bicinchoninic acid method. Then, equivalent weights of protein (40 μg/lane) were separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) buffer and then incubated with the following rabbit anti-rat polyclonal primary antibodies: α-SMA (1:1,000 dilution; Abcam; ab5694), TGF-β1 (1:1,000 dilution; Santa Cruz Biotechnology, Inc.; sc146) and Smad7 (1:1,000 dilution; Santa Cruz Biotechnology, Inc.; sc11392). Subsequently, after washing twice with PBS, the membranes were incubated with secondary antibody (ZSGB-BIO Co.) conjugated with horseradish peroxidase at 1:2,000 dilution. Specific bands were visualized using an Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, Darmstadt, Germany).
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