Cells were lysed in buffer (20 mM Tris-HCl pH 8, 150 mM NaCl) containing a protease inhibitor. Protein concentrations were determined using a BCA Protein Assay Kit. A total of 20–30 μg of proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) non-fat milk in Tris-buffered saline with 20% TWEEN-20 (TBS-T). Membranes were then incubated in primary antibodies overnight: anti-TGF-β1 (Santa Cruz Biotechnology), Anti-α-SMA (Sigma-Aldrich), Anti-collagen I (Santa Cruz Biotechnology), Anti-fibronectin (Abcam), Anti-iNOS (Abcam), Anti-IL-10 (Santa Cruz Biotechnology), Anti-p-Smad2 (Cell Signaling Technology), Anti-Smad2 (Santa Cruz Biotechnology), Anti-p-Smad3 (Cell Signaling Technology), Anti-Smad3 (Santa Cruz Biotechnology), Anti-Smad7 (Santa Cruz Biotechnology), and Anti-p-NF-κB, Anti-NF-κB, Anti-p-IκBα, Anti-IκBα (Cell Signaling Technology). After incubation, anti-rabbit IgG and anti-goat IgG (Santa Cruz Biotechnology) were used to detect proteins. The membranes were visualized using an enhanced chemiluminescence detection (ECL) kit (Amersham Pharmacia Biotech, Piscataway, NJ, United States). Densitometric analysis was performed using ImageJ software1 .
Anti smad2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Smad2 is a reagent used in research applications. It functions as an antibody that specifically binds to the Smad2 protein, which is a key component of the TGF-beta signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti smad3, by Santa Cruz Biotechnology (5 mentions)
Anti p smad3, by Santa Cruz Biotechnology (3 mentions)
Anti tgf β1, by Santa Cruz Biotechnology (2 mentions)
Anti smad7, by Santa Cruz Biotechnology (2 mentions)
Anti p smad2, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti collagen 1, by Santa Cruz Biotechnology (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
Anti inos, by Abcam (1 mentions)
Anti il 10, by Santa Cruz Biotechnology (1 mentions)
Anti p smad2, by Cell Signaling Technology (1 mentions)
Anti p smad3, by Cell Signaling Technology (1 mentions)
Anti p nf κb, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti tgf β, by Santa Cruz Biotechnology (1 mentions)
12 protocols using anti smad2
1
Western Blot Analysis of Fibrosis Markers
2
Renal Immunohistochemical Analysis
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Five-micron-thick renal sections were deparaffinized, rehydrated, and immersed in phosphate-buffered saline. Then, the sections were stained with anti-TGF-β (1:100, Santa Cruz Biotechnology, Dallas, TX, United States), anti-β-catenin (1:100, Santa Cruz Biotechnology, Dallas, TX, United States), and anti-Smad2 (1:100, Santa Cruz Biotechnology, Dallas, TX, United States) antibodies at 4°C overnight. Tissue sections were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000, Santa Cruz Biotechnology, Dallas, TX, United States) for 1 h at room temperature. Immuno-labeling was visualized with 0.05% diaminobenzidine. A digital microscope (Nikon, Tokyo, Japan) was used to analyze sections at 400 × magnification to identify positive cells.
3
Western Blot Analysis of Cellular Proteins
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Harvested cells were washed once with PBS and lysed to extract total cellular protein using RIPA buffer (Beyotime Institute of Biotechnology). The proteins were boiled for 5 min and protein concentration was quantified using a Micro-BCA protein assay. Subsequently, 20 µg/lane protein was subjected to 10% SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked in 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-ANXA1 (1:500; cat. no. AMAB90558; Sigma-Aldrich; Merck KGaA), anti-PTHrP (1:200; cat. no. sc-53936; Santa Cruz Biotechnology, Inc.), anti-Smad2 (1:500, cat. no. 5339; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-Smad2 (1:500; cat. no. 18338; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,000; cat. no. A1978; Sigma-Aldrich; Merck KGaA). Subsequently, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; OriGene Technologies, Inc.) or goat anti-mouse IgG (1:5,000; cat. no. ZB-2305; OriGene Technologies, Inc.) secondary antibodies for 1 h at room temperature. Finally, the target proteins were visualized by chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) and measured using ImageJ software (version 1.52a; National Institutes of Health).
4
Quantitative Western Blot Analysis of TGF-β Signaling
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ESCs and endometrial tissues were first lysed with lysis buffer and centrifuged at 12,000 × g for 15 min at 4°C. A BCA protein assay kit (Beyotime) was used to determine the quantity of protein. Protein samples (50 μg) were separated with SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween-20, pH 7.4) for an hour at room temperature and then incubated in primary antibody overnight at 4°C. The primary antibodies used in this study were rabbit anti-β-actin (1 : 2500, control), anti-TGF-b1 (1 : 1000), anti-p-Smad2 (1 : 500), anti-Smad2 (1 : 1000), anti-p-Smad3 (1 : 1000), anti-Smad3 (1 : 500), anti-Smad4 (1 : 200), and anti-Smad7 (1 : 500) all from Santa Cruz Biotechnology. Membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1 : 10,000) for an hour at room temperature. Protein bands were visualized using Luminata Crescendo Western HRP Substrate (Millipore, Billerica, MA, USA) and a molecular imager (Bio-Rad, Philadelphia, PA, USA). Densitometry analysis was determined relative to β-actin using 1-D Analysis Software (National Institutes of Health, USA).
5
Western Blot Analysis of TMEM88, Smad2/3 Signaling
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Tissues or cells were homogenized and lysed using RIPA lysis buffer (Beyotime, Nantong, China). Equal amount of protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a nitrocellulose membrane (Millipore). The membranes were blocked with 5% defatted milk in TBST buffer at room temperature for 1 h, and then incubated with primary antibodies overnight at 4 °C. The primary antibodies were anti-TMEM88, anti-p-Smad2, anti-Smad2, anti-p-Smad3, anti-Smad3 and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Finally, the antigen–antibody complexes were determined using an enhanced chemiluminescence (Gibco, Rockville, MD). The absorbance values of the target proteins were performed through Gel-Pro Analyzer version 4.0 software (Media Cybernetics, Silver Spring, MD, USA).
6
Protein Expression Analysis Protocol
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Cells were washed with ice-cold phosphate buffered saline and incubated in extraction buffer (20 mM Tris-Cl [pH 7.4], 100 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 5 mM MgCI2, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM pepstatin A, 0.1 mM antipain, 0.1 mM chemostatin, 0.2 mM leupeptin, 10 μg aprotinin, 0.5 mg/mL soybean trypsin inhibitor, and 1 mM benzamidine) on ice for 15 minutes. Equal amounts of total cell protein (100 μg) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide denaturing gels and transferred to nitrocellulose membranes. The blots were then probed with anti-human COX-2, anti-Smad2, and anti-Smad3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were also incubated with antibody specific for α-tubulin (Sigma Chemical Co., St. Louis, MO) as a loading control. Antibody binding was detected using an ECL system (Amersham Pharmacia Biotech, Piscataway, NJ).
7
Protein Expression Analysis in Kidney Tissues
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Cells and kidney tissues were washed with PBS and lysed in the M-PER mammalian protein extraction reagent with protease inhibitor cocktail (Thermo Fisher Scientific Inc., San Jose, CA, USA). Proteins were separated with 8–15% SDS-PAGE and then were transferred onto a nitrocellulose membrane (Millipore, Madrid, Spain) by electroblotting. The membrane was blocked for 1 hour at room temperature and then was incubated overnight at 4 °C with anti-Shh, anti-E-cadherin, anti-Smad2, anti-Smo, anti-Gli-1 (1:1000, Santa Cruz biotechnology, Santa Cruz, CA, USA), anti-fibronectin (R&D system Inc. Minneapolis, MN, USA), anti-Bax, anti-Bcl-2, anti-TGF-β1 (1:1000, Cell Signaling Technology, Beverly, MA, USA), and α-SMA (1:1000 Abcam Inc. Cambridge, MA, USA) primary antibodies. Subsequently, the membranes were stained with horseradish peroxidase-conjugated goat anti-rabbit or mouse immunoglobulin G (1:2,000, Santa Cruz biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were detected by chemiluminescence (enhanced chemiluminescence; BioFX Laboratories Inc., Owings Mills, Maryland, USA). GAPDH (1:2,000, Santa Cruz biotechnology, Santa Cruz, CA, USA) was used as an internal control.
8
Molecular Characterization of Fibrosis
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Cell lysates were subjected to SDS-PAGE and transferred to the PVDF membrane (Amersham, Arlington Heights, IL, USA) by a wet-transfer. Membranes were probed with primary antibodies overnight at 4 °C. The primary antibodies used included anti-α-SMA (Cat. #A5228), anti-COL1A1 (Cat. #C2456), anti-Smad2, and anti-phosphorylated Smad2 (Cat. #ZRB04953) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following incubation of primary antibodies, the membranes were rinsed three times and incubated with corresponding secondary antibodies. The immunoreactive bands were developed using an ECL-plus chemiluminescence substrate (Perkin-Elmer, Waltham, MA, USA) and detected by the LAS-1000 plus Luminescent Image Analyzer (GE Healthcare, Piscataway, NJ, USA).
9
Western Blot Analysis of Fibrosis Markers
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Western blot analysis was performed as previously described [46 (link)]. Membranes were probed with one of the following primary antibodies: anti-NOX-4 (PA5-72816), anti-TGFβ1 (Santa Cruz Biotechnology, Heidelberg, Germany, sc-130348), anti-p-Smad3 (cell signaling), anti-p-Smad2 (cell signaling), anti-Smad3 (cell signaling), anti-Smad2 (cell signaling), anti-Col13a1 (Santa Cruz Biotechnology, sc-514601), anti-Col11a1 (Thermo Fisher), in 1 x PBS, 0.1% Tween-20, 5% w/v non-fat dried milk (PMT) at 4 °C overnight [47 (link),48 (link)]. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) [49 (link)]. Blots were also incubated with primary antibody against GAPDH (Santa Cruz Biotechnology). Signals were detected with an enhanced chemiluminescence detection system reagent according to the manufacturer’s instructions (SuperSignalWest Pico Chemiluminescent Substrate, Pierce) [49 (link)].
10
Idazoxan and TGF-β Modulate Liver Fibrosis
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Idazoxan (IDA) and transforming growth factor-β (TGF-β) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carbon tetrachloride (CCl4) was purchased from Xilong Chemistry Plant (Shantou, China). Perifosine (PE), anti-Nrf2, anti-HO-1, anti-α-SMA and anti-Col1 were purchased from Abcam (Cambridge, UK). Anti-NQO1, anti-Smad2, anti-phospho-Smad2, anti-Smad3, anti-p-Smad3, anti-keap1, anti-GAPDH, anti-PCNA, anti-SOD2, anti-Catalase, anti-p38, anti-p-p38, anti-Akt and anti-p-Akt were obtained from Santa Cruz (CA, USA). IL-1β, IL-6, TNF-α and TGF-β enzyme-linked immunosorbent assay (ELISA) kits were obtained from Boster Biotechnology (Wuhan, China). Reactive Oxygen Species Assay Kit, BCA Protein Assay Kit, Nuclear and Cytoplasmic Extraction kits, Total Superoxide Dismutases Assay Kit, glutathione perosidase (GPx) Assay Kit and Catalase Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China).
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