Cellular proteins were isolated from hepatic tissues in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, 1 mM EDTA) (Millipore, Billerica, MA, USA) containing a protease inhibitor cocktail (Roche) and quantified with bicinchoninic acid protein concentration assay kit (Beyotime Biotech, Beijing, China). Protein of each sample was separated by electrophoresis in 10% SDS-PAGE with a Bio-Rad electrophoresis system (Hercules, CA, USA). After electrophoresis, the resolved protein was transferred onto PVDF membranes (Millipore). Membranes were then incubated with primary rabbit antibodies: α-SMA (1:1000, UCallM biotech Co., Ltd, Wuxi, China), MyD88 (1:1000, UCallM biotech Co., Ltd, Wuxi, China), p-NF-κB (1:1000, UCallM biotech Co., Ltd, Wuxi, China), TGF-β1 (1:1000, Affinity Biosciences, OH, USA), p-Smad2/3 (1:1000, Proteintech, Wuhan, China), BAMBI (1:1000, Proteintech, Wuhan, China) and β-actin (1:2000, Santa Cruz Biotechnology, CA, USA) at 4 °C overnight. The corresponding horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit IgG, Santa Cruz Biotechnology, CA, USA. 1:2000 dilutions) were incubated for 2 h at room temperature. The protein bands were visualized using enhanced chemiluminescence reagents (Millipore). The optical density of each protein in each sample was normalized against β -actin.
Tgf β1
Manufactured by Affinity Biosciences
Sourced in United States, China
TGF-β1 is a recombinant human transforming growth factor beta 1 protein. It is a cytokine that plays a crucial role in the regulation of cell growth, differentiation, and other cellular functions.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
α sma, by Proteintech (3 mentions)
E cadherin, by Affinity Biosciences (3 mentions)
P smad3, by Affinity Biosciences (3 mentions)
Vimentin, by Proteintech (2 mentions)
Col1a1, by Novus Biologicals (2 mentions)
Smad2 3, by Affinity Biosciences (2 mentions)
Tβrii, by Abcam (2 mentions)
Vimentin, by Affinity Biosciences (2 mentions)
Gapdh, by Affinity Biosciences (2 mentions)
Bicinchoninic acid protein concentration assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
P smad2 3, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Electrophoresis system, by Bio-Rad (1 mentions)
N cadherin, by Proteintech (1 mentions)
Ecl solution, by Beyotime (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bca kit, by Beyotime (1 mentions)
13 protocols using tgf β1
1
Hepatic Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis of ESCC Cells
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The ESCC cells were lysed and the overall protein was extracted. A BCA kit (Beyotime Biotechnology, China) was used to determine the concentration of the protein. Equal amount of protein was separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Then, the membranes were blocked with 5% non-fat dry milk in Tris-buffered saline Tween (TBST, pH 7.4) for 1 hr, and then incubated with primary antibodies against 12-LOX (Novus Biologicals, USA, diluted 1:700), TGF-β1 (Affinity Biosciences, China, diluted 1:1000), vimentin (CST, USA, diluted 1:1000), N-cadherin (Proteintech, USA, diluted 1:1000), E-cadherin (CST, USA, diluted 1:1000) and GAPDH (Santa Cruz Biotechnology, USA, diluted 1:2000) for 4°C overnight. Subsequently, after washed with TBST, the membranes were incubated with secondary antibody (Affinity Biosciences, China, diluted 1:5000) for 1 hr at room temperature. The membranes were washed and detected using ECL solution (Beyotime Biotechnology, China).
3
Western Blot Analysis of Fibrosis Markers
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The tissue around the material was frozen and milled in liquid nitrogen, and the cell protein was extracted with RIPA lysis buffer. After electrophoretic separation, the proteins were transferred to PVDF membranes, and the membranes were blocked with 5% skimmed milk powder solution at room temperature for 1 h. Next, the membranes were incubated with primary antibody at 4°C overnight. The membranes were washed with TBST 3 times for 10, the secondary antibody (antibody dilution ratio of 1:8,000) was added, and the membranes were incubated for 1 h. The membranes were then washed with TBST 3 times for 10 min. Luminescent reaction solution was added, and the protein expression content was determined. The primary antibodies were as follows: Smad2/3 (1:500, Affinity, United States), vimentin (1:500, Proteintech, China), α-SMA (1:500, Proteintech, China), Col-1A1 (1:500, Novus, United States), TGF-β1 (1:500, Affinity, United States), TβRI (1:500, Abcam, United States), TβRII (1:500, Abcam, United States), Smad2/3 (1:500, Affinity, United States), p-Smad2/3 (1:500, Affinity, United States), and β-actin (1:1,000, Abcam, United States).
4
Asiaticoside and TGF-β1 Fibroblast Differentiation
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Logarithmic growth phase fibroblasts were inoculated into a Millicell chamber with different materials at a density of 1 × 104 cells per well. When the cells grew to 70% confluence, they were starved with serum-free medium for 24 h. After 72 h of treatment with 500 mg/L asiaticoside or/and 10 ng/ml TGF-β1 for 72 h, the cells were washed with PBS three times, fixed with paraformaldehyde for 30 min, and permeabilized with Triton X-100 for 20 min. The cells were then blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h, and then incubated in primary antibody overnight at 4°C. The cells were washed three times with PBS and fluorescent secondary antibody (1:100, Invitrogen, United States) was added, followed by incubation at room temperature for 1 h. DAPI was applied to stain the nuclei after washing with PBS and the cells were subsequently photographed under the microscope. The primary antibodies were as follows: vimentin (1:50, Proteintech, China), α-SMA (1:20, Proteintech, China), Col-1A1 (1; 10, Novus, United States), TGF-β1 (1:200, Affinity, United States), TβRI (1:50, Abcam, United States), TβRII (1:50, Abcam, United States), and Smad2/3 (1:100, Affinity, United States).
5
Penile Tissue Immunohistochemistry: Transforming Growth Factor and Nitric Oxide Synthases
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Penile tissue sections were processed for immunohistochemical investigations and incubated with antibody against TGF‐β1 (1:100; Affinity Biosciences), eNOS (1:100; Affinity Biosciences) or nNOS (1:100; Affinity Biosciences) at 37°C for 1 hour. The sections were then incubated with biotinylated secondary antibodies. Masson's trichrome staining was carried out to determine the ratio between smooth muscle and collagen in the corpus cavernosum as previously described.26
6
Nuclear and Cytoplasmic Protein Extraction
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The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Co., Ltd. P0027) was used to extract nuclear and plasma proteins from the tissues and cells. All lysates contained protease inhibitors. The quantified proteins were separated by 10% SDS-PAGE. After transferring the protein to the PVDF membrane and blocking with 5% BSA, the PVDF membrane was blocked at room temperature with the following primary antibodies: HIF-1α (Affinity, Changzhou, China), TGFΒ1 (Affinity, Changzhou, China), SAMD3 (Affinity, Changzhou, China), p-SMAD3 (Affinity, Changzhou, China) E-cadherin (Affinity, Changzhou, China), Vimentin (Affinity, Changzhou, China), Lamin B (Affinity, Changzhou, China) and GAPDH (Affinity, Changzhou, China). GAPDH and Lamin B were used as loading controls. After 4 h, the excess primary antibody was removed, and the PVDF membrane was incubated with HRP-labelled secondary antibody at room temperature for 2 h. Protein intensity was detected with an Image Lab instrument (Bio-Rad, USA). Each experiment was performed in triplicate.
7
Inhibition of TGF-β1-Induced Fibrosis
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PPD was supplied by Chengdu Biopurify Phytochemical Ltd. (Chengdu, China) and BLM and nintedanib (NDN) by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All cell culture reagents came from Biosharp Technology Inc. (Hefei, China) and Gibco (Grand Island, NY, USA). Recombinant Mouse TGF-β1 was supplied by Suzhou Nearshore Protein Technology Co., Ltd. (Suzhou, China). PCR primers for TGF-β1, COL Ⅰ, α-SMA, STING, E-cadherin, Vimentin and β-actin were purchased from Beijing Qingke Biological Technology Co., Ltd. (Beijing, China). Control small interfering RNA, STING-siRNA and AMPK-siRNA were provided by Santa Cruz Biotechnology Inc. (Dallas, Texas, U.S.A.). Antibodies raised against Smad2, p-Smad2, AMPK, p-AMPK and TGF-β1 were purchased from Affinity Biosciences LTD. (Ohio, USA) and against α-SMA, Col I, β-actin and GAPDH from Proteintech Group Inc. (Chicago, USA).
8
Migration and Invasion Assays for Cell Lines
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For migration assays, cells were seeded in a 24-well plate at a density of 2 × 105 cells/well. After 24 h, a wound was made in the center of the well, and baicalin (50 μM, purchased from ApexBio, TX, USA) or TGF-β1 (10 ng/mL, purchased from Affinity) was added and incubated for 48 h. Then, images were taken with a microscope.
Invasion assays were performed with a 24-well plate and 8 micron Matrigel Invasion Chambers. In brief, chambers were placed into the well of the plate, and 50 μL of Matrigel:medium (1:1) was added into the chambers. After solidification of the Matrigel, a 200 μL cell suspension without 10% FBS containing approximately 8 × 104 cells and baicalin (50 μM) or TGF-β1 (10 ng/mL) was added into the upper chamber. Afterward, a 500 μL medium with 10% FBS acting as a chemo-attractant was added into the lower chamber, followed by incubation at 37 °C. After 24 h, the chambers were taken out, and the medium was abandoned. After washing thrice with 1× phosphate-buffered saline (PBS), the cells transferred through the filter membrane were fixed in 4% paraformaldehyde (precooled at 4 °C) and stained with crystal violet for 20 min at room temperature. At the end of the experiments, photographs were taken with a microscope.
Invasion assays were performed with a 24-well plate and 8 micron Matrigel Invasion Chambers. In brief, chambers were placed into the well of the plate, and 50 μL of Matrigel:medium (1:1) was added into the chambers. After solidification of the Matrigel, a 200 μL cell suspension without 10% FBS containing approximately 8 × 104 cells and baicalin (50 μM) or TGF-β1 (10 ng/mL) was added into the upper chamber. Afterward, a 500 μL medium with 10% FBS acting as a chemo-attractant was added into the lower chamber, followed by incubation at 37 °C. After 24 h, the chambers were taken out, and the medium was abandoned. After washing thrice with 1× phosphate-buffered saline (PBS), the cells transferred through the filter membrane were fixed in 4% paraformaldehyde (precooled at 4 °C) and stained with crystal violet for 20 min at room temperature. At the end of the experiments, photographs were taken with a microscope.
9
Analysis of TGF-β1 and p-mTOR in Lung Samples
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We collected sample lungs and lysed them for 5 min in buffer with 1% proteinase inhibitors on ice, and then a BCA assay kit (Wanlei Biological Technology Co., Ltd.) was used to determine protein concentrations in the supernatant. The protein (40 μg) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes, after which, PVDF membranes were blocked with 5% nonfat milk containing Tris‐buffered saline with Tween for 1 hr at room temperature. The membranes were then incubated with primary antibodies against TGF‐β1 (Affinity Biosciences) and phosphorylated (p)‐mTOR (Affinity Biosciences) overnight at 4°C and incubated with secondary antibody for 60 min at room temperature. β‐actin was used as an internal control that showed no differences between test groups. Luminescence signals were detected with an enhanced chemiluminescence system (Wanlei Biotechnology Co., Ltd.), and Gel‐Pro‐Analyzer software version 4.0 (Media Cybernetics Inc.) was used to analyze signal strength.
10
Protein Expression in Corneal Tissues
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As previously described,26 (link) corneal tissues (three corneas per sample) and cells were subjected to radioimmunoprecipitation assay buffer lysis to extract the total protein. Thereafter, the protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking the membranes with 5% skim milk for 1 hour, they were incubated with primary antibodies against NLRP3 (1:1000; Adipogen Life Sciences, San Diego, CA), ASC (1:1000; Abcam), IL-1β (1:1000; Abcam), caspase 1 (1:1000; Abclonal, Wuhan, China), α-SMA (1:1000, Abcam), TGF-β1 (1:1000, Affinity Biosciences, Cincinnati, OH), and β-actin (1:1000, Affinity Biosciences). Subsequently, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:1000; Zhongshan Jinqiao Biotech, Beijing, China). Finally, the proteins were visualized using an automatic chemiluminescence image analysis system (Tanon, Shanghai, China), and the expression levels of the target proteins were analyzed using ImageJ software with β-actin as an internal control. The IL-1β levels in the supernatants of the macrophage suspensions were analyzed using commercially available murine IL-1β ELISA kits (BOSTER, Pleasanton, CA), according to the manufacturer's instructions.
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