Placental tissues were homogenized in lysis buffer with added proteinase inhibitors (Roche, Penzberg, Germany). A BCA kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to assess protein concentrations. 20 ug of proteins were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Applied Biosystems, Waltham, Massachusetts, USA). The membranes were incubated with the primary antibodies against TGF-β2 (Abcam, Cambridge, UK, 1:500) and GAPDH (Abmart, Shanghai, China, 1:1000) overnight at 4 °C. Following this, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Abmart, Shanghai, China, 1:3000). The signal intensity was quantitatively assessed with Fujifilm LAS-3000 Imager and was normalized to GAPDH.
Tgf β2
Manufactured by Abcam
Sourced in United States, United Kingdom
TGF-β2 is a member of the transforming growth factor beta (TGF-β) family of cytokines. It is a secreted protein that plays a role in the regulation of cell growth, proliferation, and differentiation.
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Lab products found in correlation
Tgf β1, by Abcam (3 mentions)
Tgf β3, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Runx2, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Gapdh, by Abmart (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Las 3000 imager, by Fujifilm (1 mentions)
Tia1 antibody, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by Beyotime (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti p smad2, by Abcam (1 mentions)
P smad3, by Abcam (1 mentions)
Smad2, by Abcam (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Smad3, by Abcam (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (1 mentions)
20 protocols using tgf β2
1
Western Blot Analysis of Placental TGF-β2
2
Western Blot Analysis of Cellular Proteins
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The method used for Western blot analysis has been described in our previous study (9 (link)). The primary antibodies were TIA1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TGFβ2 (Abcam, Cambridge, MA, USA) and β-actin antibody (Beyotime, Nantong, China).
3
Bacterial Taxis Regulated by Lung Cancer Proteins
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According to the proteomic analysis results, important candidates of lung cancer regulation, clusterin (CLU, 2 ug ml−1, proteintech, Wuhan, China), serglycin (SRGN, 2 ug ml−1, proteintech, Wuhan, China), transforming growth factor- β2 (TGFβ2, 2 ug ml−1, Abcam, England, UK) were examined for taxis of bacteria. The PBS and BME were poured in both of the cell culture chambers whereas candidate proteins were added only in one of the chambers.
4
Western Blot Analysis of Signaling Proteins
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Tissues or cells were lysed using ice-cold lysis buffer (50 mM Tris—HCl, pH7.0, 1%w/v SDS, 10%glycerol) and were centrifugated at 4°C. Subsequently, proteins in the supernatants were quantified. After being separated by10% SDS PAGE, the proteins were blotted onto nitrocellulose membrane (Amersham BioSciences, Buckinghamshire, UK). After being blocked with 10% nonfat milk in PBS for 1 hour, the membranes were immunoblotted with antibodies as indicated. Then the membranes were immunoblotted by HRP-linked secondary antibodies (Cell Signaling). The signals were detected by SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA). Anti-p-SMAD2, SMAD2, p-SMAD3, SMAD3, EGFR, TGF-β2 and IGFBP-3, and glyceraldehydes 3-phosphate dehydrogenate (GAPDH) antibodies were obtained from Abcam Company (USA). Enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) system was used to visualize intensity of the bands, which were subsequently exposed.
5
Western Blot Analysis of β-catenin and TGFβ2
6
Protein Expression and Signaling Pathway Analysis
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In brief, the cells were harvested, and lysed with protein extraction agent (Beyotime, Beijing, China). 25–50 μg of proteins per sample per lane were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Primary antibodies were incubated overnight at 4 °C. Rabbit polyclonal primary antibodies were used against LC3B, SQSTM1, MMP2, MMP9, vimentin, N-cadherin, β-catenin, Slug2, Akt, JNK, SMAD2 (1:1000; Cell Signaling), TGF-β2 (1:500; Abcam), phospho-SMAD2, phospho-Akt, and phospho-JNK (1:500; Cell Signaling). Purified mouse anti-β-actin was used (1:1000; Santa Cruz Biotechnology, Inc.). Secondary antibodies were incubated with anti-mouse immunoglobulin G (IgG), anti-rabbit IgG (1:5000; Santa Cruz Biotechnology) for 1 h at RT. The proteins were visualized using Millipore’s enhanced chemiluminescence (ECL) and detection system analyzed (ChemiDoc Touch, BioRad).
7
Protein Expression Analysis in Rat Liver
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The rat livers were lysed in RIPA buffer (RIPA Lysis and Extraction Buffer, Thermo Scientific Pierce, USA; Cat# 89900), and the protein extracts were measured using a Bradford Assay Kit (Thermo Scientific Pierce, USA; Cat# 23200). Equal amounts of protein (50 μg) were heat denatured in 4× sample buffer (2% sodium dodecyl sulfate, 62.5 mM Tris (pH 6.8), 0.01% bromophenol blue, 1.43 mM mercaptoethanol, and 0.1% glycerol), separated on a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel, and electroblotted onto a nitrocellulose membrane (Thermo Scientific Pierce, USA; Cat# 88025, Shanghai, China). The membranes were subsequently treated with the appropriate antibodies against the following proteins: TGF-β1 (1/1000) (Cat# ab179695, Abcam, Shanghai, China), TGF-β2 (1/500) (Cat# 167655, Abcam, Shanghai, China), TNF-α (1/1000) (Cat# ab66579, Abcam, Shanghai, China), UCP-2 (1/500) (Cat# ab67241, Abcam, Shanghai, China), PPAR-α (1/1500) (Cat# GTX101098, GeneTex, Shanghai, China), PPAR-γ (1/2000) (Cat# ab191407, Abcam, Shanghai, China), LXR-α (1/1000) (Cat# ab41902, Abcam, Shanghai, China), ELOVL-2 (1/5000) (Cat# ab176327, Abcam, Shanghai, China) and β-actin (1/10000) (Cat# a5441, Sigma-Aldrich, Shanghai, China).
8
Histological Analysis of Hydrogel Samples
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The hydrogels were fixed in 4% paraformaldehyde for 24 h, embedded in OCT compound, and processed using standard procedures. For the immunofluorescence microscopy, 20-µm-thick histological sections were used, which were fixed for 10 min with 4% paraformaldehyde in PBS at room temperature, washed three times with 1 × PBS, and permeabilized with 0.5% Triton-X for 10 min. Sections were washed with PBS and blocked for 45 min in blocking buffer (5% BSA and 0.5% Tween-20 in 1× PBS) containing 10% normal goat serum at room temperature. For immunostaining, sections were incubated overnight at 4 °C with collagen 10 (1:200, Sigma), RUNX2 (1:200, Abcam, Cambridge, UK), chondroitin sulfate (1:100, Abcam), TGF-β2 (1:200, Abcam), and DKK1 (1:200, Abcam). The secondary antibodies were goat anti-rabbit Alexa Fluor 568 and goat anti-mouse Alexa Fluor 488 (Abcam), which were incubated for 1 h at room temperature. Images were acquired using the Cytation 3 Cell Imaging Multi-Mode Reader (Biotek Instruments, Inc., Winooski, VT, USA). An automated detection of the fluorescent intensities was used to analyze the expression.
9
Western Blot Analysis of Cardiac Protein Expression
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Protein expression levels were determined by western blot analysis as previously described.26 (link)
Briefly, 10 μg of proteins extracted from cardiac tissues were electrophoresed in polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After 1-hour incubation with a blocking reagent, the membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibody for 1 hour at room temperature. The following antibodies were used for western blot analyses: TGF-β2 (Abcam Japan, Chuo, Tokyo, Japan; Product ID: ab36495, RRID: AB_778343; mouse monoclonal antibody; dilution: 1:2000; molecular weight [MW]: 25 kDa [bioactive dimer]), NHE-1 (Santa Cruz Biotechnology, Dallas, TX, USA; Product ID: sc-136239, RRID: AB_2191254; mouse monoclonal antibody; dilution: 1:500; MW: 110 kDa), β-actin (Santa Cruz Biotechnology; Product ID: sc-47778; RRID: AB_2714189; mouse monoclonal antibody; dilution: 1:10,000; MW: 43 kDa), and anti-mouse IgG from sheep (GE Healthcare Japan, Hino, Tokyo, Japan; Product ID: NA931; RRID: AB_ 772212; dilution: 1:20,000). The bands on immunoblots were detected using the Amersham ECL Prime Kit (GE Health Care Japan), digitized using a WSE-6100 LuminoGraph (ATTO, Taito, Tokyo, Japan), and quantified using CS Analyzer 4 software (ATTO).26 (link)
Briefly, 10 μg of proteins extracted from cardiac tissues were electrophoresed in polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After 1-hour incubation with a blocking reagent, the membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibody for 1 hour at room temperature. The following antibodies were used for western blot analyses: TGF-β2 (Abcam Japan, Chuo, Tokyo, Japan; Product ID: ab36495, RRID: AB_778343; mouse monoclonal antibody; dilution: 1:2000; molecular weight [MW]: 25 kDa [bioactive dimer]), NHE-1 (Santa Cruz Biotechnology, Dallas, TX, USA; Product ID: sc-136239, RRID: AB_2191254; mouse monoclonal antibody; dilution: 1:500; MW: 110 kDa), β-actin (Santa Cruz Biotechnology; Product ID: sc-47778; RRID: AB_2714189; mouse monoclonal antibody; dilution: 1:10,000; MW: 43 kDa), and anti-mouse IgG from sheep (GE Healthcare Japan, Hino, Tokyo, Japan; Product ID: NA931; RRID: AB_ 772212; dilution: 1:20,000). The bands on immunoblots were detected using the Amersham ECL Prime Kit (GE Health Care Japan), digitized using a WSE-6100 LuminoGraph (ATTO, Taito, Tokyo, Japan), and quantified using CS Analyzer 4 software (ATTO).26 (link)
10
Protein Expression Analysis by Western Blot
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Total proteins were extracted from the cells using RIPA buffer with PMSF (Beyotime Institute of Biotechnology) on ice, subjected to SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated in 5 % nonfat milk dissolved in Tris-buffered saline (TBS) containing 0.1 % Tween-20 for 1.5 h at room temperature and then incubated with primary antibodies as follows: TGF-β2 (1:1000, Abcam, EUGENE, USA), β-actin (1:1000, Santa Cruz Biotechnology). After incubation with secondary antibodies (Goat anti-rabbit or Goat anti-mouse, 1:5000 respectively; ZSGB-BIO, Beijing, China), immune complexes were visualized by SuperSignal® West Femto Trial Kit (Thermo Fisher, USA) and blot bands were scanned using Find-do × 6 Tanon (Tanon, Shanghai, China).
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