Proteins were extracted by radioimmunoprecipitation assay (RIPA) Lysis buffer (Abcam, Cambridge, MA, USA) and quantified by bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). 40 µg of protein per sample was separated onto 10% SDS-PAGE gels and transferred onto polyvinyl difluoride (PVDF) filter membrane (Bio-Rad, Hercules, CA, USA). Then, PVDF membranes were blocked with 5% non-fat dried milk for 2 h, and incubated with anti-BZW1 (1: 1000, ABcam Corp, USA), anti-Smad1 (1: 1000, ABcam Corp, USA), anti-p-Smad1 (1: 1000, ABcam Corp, USA), anti-Smad3 (1: 1000, ABcam Corp, USA), anti-p-Smad3 (1: 1000, ABcam Corp, USA), anti-TGF-β1 (1: 2000, ABcam Corp, USA) and anti-GAPDH (1: 2000, ABcam Corp, USA), respectively at 4 overnight. Finally, membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-rabbit). Signals were developed using an enhanced chemiluminescence reaction kit (Applygen Technologies, Beijing, China).
Anti tgf β1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-TGF-β1 is a lab equipment product that functions as an antibody specific to transforming growth factor beta 1 (TGF-β1). This antibody can be used for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma, by Abcam (10 mentions)
Anti smad3, by Abcam (8 mentions)
Anti p smad3, by Abcam (6 mentions)
Anti gapdh, by Abcam (5 mentions)
Anti ctgf, by Abcam (4 mentions)
Anti collagen 1, by Abcam (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti smad2, by Abcam (3 mentions)
Anti tnf α, by Abcam (3 mentions)
Anti erk1 2, by Cell Signaling Technology (3 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti bax, by Abcam (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti at1r, by Abcam (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti ctnt, by Abcam (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
57 protocols using anti tgf β1
1
Western Blot Analysis of BZW1, Smad, and TGF-β1
2
Gumiganghwal-tang Extraction Protocol
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The chopped crude herb of Gumiganghwal-tang was purchased from Omniherb (Yeongcheon and Kunwi, Korea) and HMAX (Jeongseon, Korea and China), and GGTA was prepared from a mixture of herbs in our laboratory (Table 1 ). GGTA was obtained via extraction in distilled water at 100°C for 120 min. The extract was evaporated to dryness and freeze dried (yield, 22.80%). The extracted Gumiganghwal-tang powder was stored at 4°C. Montelukast (Mon) was purchased from Sigma-Aldrich (SML0101; St. Louis, MO, USA) and was used as the positive control drug. Other reagents were as follows: ovalbumin (OVA, albumin from chicken egg white; A5503; Sigma-Aldrich), aluminum hydroxide (239186; Sigma-Aldrich), pentobarbital (Hanlim Pharm. Co., Seoul, Korea), enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA; BioSource International, Camarillo, CA; and BioLegend Corp., San Diego, CA, USA), hematoxylin solution (MHS16; Sigma-Aldrich), eosin Y solution, (HT110132, Sigma-Aldrich), and anti-TGF-β1 (ab64715; Abcam, Cambridge, UK & Cambridge, MA, USA), anti-Smad-3 (sc-101154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (#4967S; Cell Signaling Technology, Danvers, MA, USA) antibodies.
3
Quantifying Scar Tissue Composition
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Scar tissue sections were incubated at 4℃ overnight with one of the following primary antibodies: mouse anti-collagen type-I (ab6308; Abcam, Ltd., Cambridge, UK), mouse anti-collagen type-III (C7805; Sigma-Aldrich Co.), mouse anti-elastin (E4013; Sigma-Aldrich Co.), mouse anti-fibronectin (sc-52331; Santa Cruz Biotechnology), rabbit anti-transforming growth factor-β1 (anti-TGF-β1) (ab9758; Abcam, Ltd.), or mouse antinatural killer-1.1 (NK-1.1) antibody (108701; Biolegend, San Diego, CA, USA). Tissues were then incubated at room temperature for 20 min with the DAKO Envision™ Kit (DAKO, Glostrup, Denmark) as a secondary antibody. Expression levels of type-I and type-III collagens, elastin, fibronectin, TGF-β1, and NK-1.1 were semi-quantitatively analyzed with MetaMorph® image analysis software (Universal Image Corp., Buckinghamshire, UK). Results are expressed as the mean optical density of six different digital images. Immunohistochemistry was performed by the same individual who was blinded to the experimental group, and the tissue sections for analysis were obtained from the center areas of scar tissue.
4
Tissue Staining and Cytokine Analysis
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A Masson’s trichrome stain kit was purchased from Solarbio (Shanghai, China). Neutral balsam, a hematoxylin and eosin (H&E) staining system, and a rabbit anti-sheep immunohistochemistry (IHC) kit were obtained from Auragene (Hunan, China). Rat TGF-β1 and rat IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai Enzyme-linked Biotechnology (Shanghai, China). Primary anti-IL-6, anti-TGF-β1, anti-Smad 2, anti-COLl, anti-ER, and anti-PR antibodies, and secondary goat anti-rabbit antibodies were purchased from Abcam (Cambridge, MA, USA). C. albicans was obtained from the Institute of Microbiology, Chinese Academy of Sciences (Beijing, China), and cultured according to the instructions.
5
Protein Expression Analysis in Lung Tissue
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Western blot analysis was employed to detect the protein expression level of TGF-β1, CTGF, and α-SMA in lung tissue, and collagen type III, α-SMA, and AdipoR1/R2 in human lung WI-38 fibroblasts. Whole lung tissue homogenate or WI-38 lung fibroblasts lysates were prepared via lysis buffer [TianGen Biotech (Beijing) Co., Ltd.]. The lung tissue homogenate or cell lysates were separated by electrophoresis on 10% SDS-PAGE. Gels were transferred to a nitrocellulose membrane, blocked in 5% nonfat dry milk diluted in Tris-buffered saline. Blots were incubated overnight with anti-TGF-β1, anti-CTGF, anti-α-SMA, anti-collagen type III, anti-AdipoR1, or anti-AdipoR2 antibodies (all from Abcam), at 4°C. Washed blots were incubated at room temperature for 60 minutes with a secondary biotinylated antibody (Zhongshan Golden Bridge Bio-technology, Beijing). After three 10 minute washings with TBST, membranes were analyzed by Western (Bio-Rad Laboratories Inc., Hercules, CA). Sample loadings were normalized by Western blot analysis with anti-GAPDH (Abcam).
6
Immunohistochemical Analysis of Angptl2 and TGF-β1 in Tissues
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LF tissue samples were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned. After the sections were pre-treated with Target Retrieval Solution, pH 9 (Tris/EDTA buffer, pH 9; Dako Japan, Co., Ltd., Tokyo, Japan), endogenous peroxidases were blocked using periodic acid (Nichirei, Tokyo, Japan). We used the following antibodies as primary antibodies: anti-human Angptl2 antibody [19] (link), anti-vimentin, anti-CD3, anti-CD15, anti-CD20, anti-CD68 (Dako Japan), anti-S100A4 (Abcam, Cambridge, UK), and anti-human p-Smad3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After treatment using EnVision + System-HRP-labeled Polymer (Dako Japan), the labeling was visualized using a Histofine 3,3′-diaminobenzidine (DAB) kit (Nichirei). For double immunofluorescent staining, anti-vimentin (Dako Japan) and anti-Angptl2 or anti-TGF-β1 (Abcam) were used as the primary antibodies, and Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 594-labeled anti-mouse IgG (Life Technologies) were used as the secondary antibodies. Nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole (DAPI).
7
Synthesis and Characterization of Telmisartan-Labeled Probes
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2-Cl-trityl chloride resin (loading: 0.939 mmol/g), fmoc-amino acids and o-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU) were bought from GL Biochem (Shanghai). 4-Chloro-7-nitrobenzol-2-oxa-1, 3-diazole (NBD-Cl) and telmisartan were purchased from Sigma-Aldrich (USA). Cy5.5 NHS ester was obtained from APExBIO (USA). Recombinant MAS1 Protein (2 µg) was obtained from Abnova (China). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), trypsin, and Penicillin-streptomycin were obtained from Thermo Fisher (USA). DHE fluorescence probe, Apoptosis Analysis Kit, DAPI, and Hoechst 33342 dye were purchased from Molecular Probes (USA). CCK-8 kit was purchased from Dojindo Co. Ltd (Japan). The primary antibodies of anti-AT1R, anti-α-SMA, anti-Bax, anti-Bcl-2, anti-Histone 3, anti-Collagen I, anti-TGF-β1 and anti-cTnT were obtained from Abcam (Britain). The antibodies of anti-AKT, anti-p-AKT, anti-P65, anti-Caspase3 were purchased from Cell Signaling Technology (USA). ELISA kit for IL-6, TNF-α, CRE, ALT, TGF-β1 were bought from MSKBIO (China). All other solvents and reagents were commercially available and used without further purification, unless noted otherwise.
8
Quantitative Analysis of Chondrocyte-Specific Proteins
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The proteins were dissociated and separated by SDS/PAGE, and then transferred to PVDF membranes, which were incubated with primary antibodies. The following primary antibodies were used: rabbit anti-SOX9, anti-MMP2, anti-MMP3, anti-MMP9, anti-MMP14, anti-BMP4, anti-TGFβ1, anti-Smad2, anti-Smad3, anti-p-Smad2 (phosphor S255), anti-p-Smad3 (phosphor S423 + S425) and mouse anti-COL10A1 (Abcam, Cambridge, MA, USA); rabbit anti-Beta-catenin, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail, anti-Slug, and anti- GAPDH (Cell Signaling Technology, Danvers, MA, USA). Antigen-antibody complexes were detected using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) with ECL western blot detection reagent (Merck Millipore). Protein expression was quantified using US National Institutes of Health (NIH) ImageJ software.
9
Sprague-Dawley Rat Tissue Analysis
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PrimeScript™ RT Master Mix kit was bought from TaKaRa Co. (Kyoto, Japan). The cell lysis kit, RNA extraction kit, and anti-GAPDH were purchased from Sangon Biotech (China). Anti-TGF-β1, anti-CTGF, and anti-TIMP-1 were obtained from Abcam (United Kingdom). Anti-Smad 2 and anti-Smad 3 were obtained from CST (USA). Anti-MMP-1 was obtained from Thermo Fisher Scientific (USA).
Sprague-Dawley (SD) rats (male, 180–220 g) were obtained from Beijing Vital River Laboratories Animal Technology Co., Ltd. Experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institute Ethics Committee (TCM-2019-013-E04, Tianjin University of Traditional Chinese Medicine).
Sprague-Dawley (SD) rats (male, 180–220 g) were obtained from Beijing Vital River Laboratories Animal Technology Co., Ltd. Experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institute Ethics Committee (TCM-2019-013-E04, Tianjin University of Traditional Chinese Medicine).
10
Immune Protein Expression Analysis
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After treatment with recombinant human IFN-γ and TNF-α (Peprotech) for 24 h, the cells were lysed in RIPA buffer (Thermo Fisher Scientific) with protease inhibitor. Protein concentrations were quantified using BCA assay kit (Thermo Fisher Scientific). Equal amounts of protein were separated by 10% SDS-polyacrylamide gel and analyzed with the following human antibodies: anti-IDO (Merk millipore), anti-TGF-β1 (Abcam), anti-NOS2 (Santa cruz Biotechnology, Dallas, TX, USA), anti-TSG-6 (R&D systems, Minneapolis, MN, USA), and anti-GAPDH (GeneTex, Irvine, CA, USA). The proteins were detected using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific) and luminescent image analysis LAS-3000 system (Fujifilm, Tokyo, Japan).
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