Western blot analysis was performed according to a standard protocol.44 (link) The primary antibodies included anti-ACP5 (Gentex, CA, USA, 1:1,000), anti-fibronectin, anti-E-cadherin, anti-vimentin (Proteintech, Wuhan, China, 1:1,000), anti-p38, anti-ERK, anti-p-ERK, anti-AKT, anti-β-catenin, anti-SMAD2/3, anti-p-SMAD2, anti-p-SMAD3, anti-p53, anti-p53 (Ser392), anti-ubiquitin (Cell Signaling Technology, Danvers, MA, USA, 1:1,000), anti-GAPDH, and anti-β-actin (Abcam, Cambridge, MA, USA, 1:3,000) antibodies. Detection was performed using a chemiluminescent substrate system (Bio-Rad, Hercules, CA, USA). The gray values were analyzed with ImageJ software.
Chemiluminescent substrate system
Manufactured by Bio-Rad
Sourced in United States
The Chemiluminescent substrate system is a laboratory equipment designed for detecting and quantifying proteins in Western blot analysis. It generates a luminescent signal in the presence of the target protein, which can be captured and analyzed using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Anti fibronectin, by Proteintech (3 mentions)
Anti β actin, by Abcam (2 mentions)
Anti β catenin, by Cell Signaling Technology (2 mentions)
Anti acp5, by Gentex (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti ubiquitin, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti p smad2, by Cell Signaling Technology (1 mentions)
Anti p smad3, by Cell Signaling Technology (1 mentions)
Anti collagen 1, by Proteintech (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
α sma, by Abcam (1 mentions)
6 protocols using chemiluminescent substrate system
1
Western Blot Analysis of Cell Signaling
2
Protein Extraction and Western Blot
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Total cellular protein was extracted by RIPA lysis (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, PH7.4) buffer supplemented with phenylmethylsulfonyl fluoride, cocktail, and phosphorylation protease inhibitor. Cell lysis was centrifuged at 12,000 rpm for 15 min, then the supernatants were collected for determination of the protein concentration by BCA assay. All steps were performed at 4°C. 20 μg of protein were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by western blotting. The signals were detected with a chemiluminescent substrate system (Bio-Rad, Hercules, CA, United States). Relative expressions of target proteins were quantified by Image J software.
3
Protein Extraction and Western Blot Analysis
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Total cellular protein was extracted by RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, PH7.4) supplemented with phenylmethylsulfonyl fluoride, cocktail, and phosphorylation protease inhibitor. Cell lysis was centrifuged at 12,000 rpm for 15 min and the supernatants were collected for determination of the protein concentration by BCA assay. All steps were performed at 4 °C. Then 50 μg of protein were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by western blotting. The signals were detected using a chemiluminescent substrate system (Bio-Rad, Hercules, CA, USA). Relative expressions of target proteins were quantified by Image J software.
4
Western Blot Analysis of Cellular Proteins
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Cellular and lung tissue lysates were prepared using RIPA lysis buffer (Beyotime, Shanghai, China). Western blot was conducted following established protocols.20 (link) In brief, protein samples were separated through 10% SDS‒PAGE and transferred onto 0.4 μm PVDF membrane. After blocking with 5% milk for 1 hour, the membrane was exposed to anti-Bcar3 (1:1000, Boster, CA, USA), anti-Fibronectin, anti-Collagen 1, anti-GAPDH, and anti-β-actin (1:1000, Proteintech, Wuhan, China) at 4 °C overnight. Horseradish peroxidase (HRP)-labelled anti-rabbit (1:10000, Abbkine, CA, USA) or anti-mouse IgG (1:10000, Abbkine, CA, USA) secondary antibodies were then applied, followed by incubation for 1 hour. Protein bands were examined using a chemiluminescent substrate system (Bio-Rad Laboratories, CA, USA). The gray value was quantified using ImageJ software.
5
Western Blot Protein Analysis Protocol
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Western blot analysis was performed as described previously35 (link). The primary antibodies used in western blot were: Kindlin-2 (Proteintech, 1:1000 dilution), Fibronectin(FN) (Proteintech, 1:1000 dilution), Col1A1 (Boster, 1:400 dilution), α-SMA (Abcam, 1:100 dilution), Smad2/3 (Cell Signaling Technology, 1:1000 dilution), p-Smad2 (Cell Signaling Technology, 1:1000 dilution), p-Smad3 (Cell Signaling Technology, 1:1000 dilution), GAPDH (Abacm, 1:3000 dilution), β-actin (Abacm, 1:3000 dilution), and tubulin (Abcam, 1:3000 dilution). Detection was performed using a chemiluminescent substrate system (Bio-Rad). The gray values were analyzed with ImageJ software.
6
Subcellular Proteome Analysis of Lung Tissues
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Lung tissues and fibroblasts were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China). For subcellular fractionation, the protein was extracted using NE-PERTM Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific, USA). Western blot was performed according to previously reported protocols48 (link). The primary antibodies used were anti-ACP5 (Gentex, CA, USA, 1:1,000); anti-LAMIN B1, anti-FIBRONECTIN and anti-COL1A1 (Proteintech, Wuhan, China, 1:1,000); anti-α-SMA, antiphospho-β-CATENIN (Ser33/37/Thr41, Cell Signaling Technology, MA, USA, 1: 1000), and anti-β-CATENIN (Cell Signaling Technology, MA, USA, 1: 1000); and anti-β-ACTIN (Abcam, MA, USA, 1:3000). β-ACTIN bands were used as the loading control for cytoplasmic or total proteins, and LAMIN B1 bands were used as the loading control for nuclear proteins. Detection was performed using a chemiluminescent substrate system (Bio-Rad Laboratories, CA, USA). The gray values were analyzed with ImageJ software.
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