Cells (5 × 10 [5 ] cells per well) were plated in six-well plates. The following day, the cells were treated with the drug or an equal volume of DMSO for the indicated times, and lysed in 2 × lysis buffer as described previously [53 ]. Cell lysates with equal amounts of protein were separated by SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes were blocked for 1 h in blocking buffer (1 × TBS, 5% milk, 0.1% Tween 20) and placed in primary antibody diluted in 1 × TBS, 5% bovine serum albumin (BSA), 0.1% Tween 20, overnight at 4°C. The following day, the membranes were washed three times in wash buffer (0.1% Tween 20, 1 × TBS). The primary antibody was detected using horseradish-peroxidase-linked secondary antibodies, and visualized with an enhanced chemiluminescent detection system (Amersham Biosciences, Pittsburgh, PA, USA). The immunoblotting experiments were performed at least three times.
Enhanced chemiluminescent detection system
Manufactured by Cytiva
Sourced in United States, United Kingdom
The Enhanced chemiluminescent detection system is a laboratory equipment designed to perform sensitive and quantitative detection of proteins and nucleic acids. The system utilizes chemiluminescent technology to generate and capture light signals, enabling the visualization and analysis of target biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Dystrophin antibody, by Abcam (2 mentions)
Gradient polyacrylamide gel, by Bio-Rad (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Imagequant software, by GE Healthcare (2 mentions)
Mab3626, by R&D Systems (1 mentions)
Haf005, by R&D Systems (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor cocktail tablet, by Roche (1 mentions)
Anti ryr2, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Anti troponin 1, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Cw0102, by CWBIO (1 mentions)
Image pro plus version 5, by Media Cybernetics (1 mentions)
12 protocols using enhanced chemiluminescent detection system
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of IL-33 in Mouse Intestine
3
Protein Expression Analysis in Cardiac Tissue
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Frozen LV was homogenized in RIPA buffer (Millipore, Schwalbach am Taunus, Germany) containing protease and phosphatase inhibitor cocktail tablets (Roche, Mannheim, Germany). Extracted proteins (20 µg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose or Polyvinylidene Difluoride (PVDF) membranes (Bio-Rad, München, Germany). Membranes were blocked for 1 h with 5% milk in TBS-Tween at room temperature and then incubated with the following primary antibodies: Rabbit polyclonal anti-phospho-Ser2814-RyR2 (Badrilla, Leeds, UK), anti-phospho-Thr-17 Phospholamban (Badrilla), anti-RyR2 (Sigma-Aldrich, Munich, Germany), anti-CaMKIIγ and anti-CaMKIIδ (Thermo Fisher, Bonn, Germany), and mouse monoclonal anti-phospho-CaMKII (Affinity BioReagents, Golden, CO, USA), anti-Phospholamban (Millipore, Hamburg, Germany), anti-Serca2a (Affinity BioReagents), and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and finally were detected using an enhanced chemiluminescent detection system (Amersham Bioscience, Braunschweig, Germany) according to the manufacturer’s instructions. For quantification, band intensity was normalized to total protein load obtained from Ponceau-stained blot using Image Lab software (Bio-Rad).
4
Western Blot Analysis of Connexin 43 Expression
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Smooth muscle cells from the four groups were separated, and total proteins were isolated from the smooth muscle cells. Equal amounts (20 µg) of protein samples were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked in 5% milk in Tris-buffered saline and 0.1% Tween 20 (TBS-T) for 1 hour and then incubated with primary antibody to Cx43 (1:200; USCN Life Science, Wuhan, China) or β-actin (1:200; USCN Life Science) as an internal control overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10,000; USCN Life Science) for 1 hour at 25°C. After washing three times with TBS, the membrane was developed using an enhanced chemiluminescent detection system (Amersham Biosciences, Piscataway, New Jersey, USA). Signal intensities were quantitated using Quantity One software (Bio-Rad, Hercules, California, USA). Cx43 expression was quantitated by the Cx43/β-actin optical absorption ratio.
5
Detecting Dystrophin Expression in Myoblasts
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Myoblasts were treated with CRISPR-Gold following the procedure described in S10. After differentiation, cells were harvested and protein extracts for Western blot analysis were made following the procedure of Lu QL, et al., 200514 . Briefly, cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.5% deoxycholate, and 1% Nonidet P-40) containing proteinase inhibitor, and the total protein concentration was determined using the BCA protein assay kit (Thermo Scientific, USA). 150 µg of protein per sample was loaded onto a 4–20% gradient polyacrylamide gel (Bio-Rad, CA, Cat # 4561094), and run 6 hours at 35 volts, the protein content of the gel was then transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk in Tris Buffered Saline (TBS) plus 0.1% Tween20 followed by an overnight incubation with dystrophin antibody (dilution 1 : 200, Abcam, UK) in TBS plus 0.1% Tween20 to detect dystrophin. GAPDH (dilution 1 : 2000, Thermo Scientific, USA) was used as a sample loading control. Blots were washed with 0.2% Tween-20 in TBS 3 times, and then incubated with a horseradish peroxidase (HRP)-coupled secondary antibody (Azures Biosystems, USA). Antibody binding was detected using an enhanced chemiluminescent detection system (Amersham).
6
Quantitative Western Blot Analysis
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As we described previously [11] (link), protein extracts from PC3 and DU145 cells were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes (100 V for 1.5 h), and immunoreacted with a rabbit anti-human NAMPT polyclonal antibody (1:10,000, Bethyl Laboratories, Inc, Cat #A300-A375A, Montgomery, TX), or mouse anti-human β-actin monoclonal antibody. Immunoreactive proteins were detected with the enhanced chemiluminescent detection system according to the manufacturer's directions (Amersham, Little Chalfont, UK). Intensities of immunoreactive protein bands were quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). All experiments were repeated a minimum of three times.
7
Western Blot Analysis of Cardiac Proteins
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Harvested left ventricle were homogenized in RIPA buffer (Millipore) containing protease and phosphatase inhibitors (Roche Molecular Biochemicals). Twenty μg protein lysates were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis. Western blotting was carried out according to standard protocols, using rabbit polyclonal anti‐PDE4DIP (Sigma‐Aldrich), anti‐phospho‐Ser16‐phospholamban (PLN, Badrilla), anti‐phospho‐Ser23/24 Troponin‐I, anti‐Troponin‐I (Cell Signalling Technology) and mouse monoclonal anti‐PLN (Millipore), anti‐GAPDH (Santa Cruz Biotechnology) as primary, and horseradish peroxidase conjugated donkey anti‐rabbit and sheep anti‐mouse immunoglobulin G (Amersham Bioscience) as secondary antibodies. Signals were quantified using enhanced chemiluminescent detection system (Amersham Bioscience) according to the manufacturer's instructions.
8
Protein Expression Analysis in Cell Lysates
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Total proteins were extracted from cells using RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA). After determining protein concentration, 20 μg of total protein from each sample was separated with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk and incubated with anti-human c-Myc (dilution, 1 : 300; 10057-1-AP, Proteintech, Chicago, IL, USA), Bcl-2 (dilution, 1 : 200; 2870, Cell Signaling Technology, USA), Bax (dilution, 1 : 200; BS2538, Bioworld, USA), cyclin-D1 (dilution, 1 : 500; BS1741, Bioworld, USA), and Glut1 (dilution, 1 : 1000; FO6231163, Wanleibio, Shenyang, China) antibodies overnight at 4°C and then incubated with goat anti-mouse (dilution, 1 : 2000; CW0102, CWBIO, Beijing, China) and goat anti-rabbit (dilution, 1 : 2000; CW0103, CWBIO, Beijing, China) secondary antibodies for 1 hour at 37°C. The blots were visualized using the enhanced chemiluminescent detection system (Amersham, Amersham, UK) and analyzed using Image-Pro Plus version 5.1 (Media Cybernetics Inc., Rockville, MD, USA).
9
Detecting Dystrophin Expression in Myoblasts
10
Western Blot Analysis of Phosphorylated NF-κB
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As we described previously [12 (link)], protein extracts from PC3 and DU145 cells were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes (100 V for 1.5 h), and immunoreacted with a rabbit anti-human phosphorylated-NFκB polyclonal antibody (1:1000, Bethyl Laboratories, Inc, Montgomery, TX, USA), total-NFκB (1:1000, Bethyl Laboratories, Inc, Montgomery, TX, USA) or mouse anti-human β-actin monoclonal antibody (1:1000, Bethyl Laboratories, Inc, Montgomery, TX, USA). Immunoreactive proteins were detected with the enhanced chemiluminescent detection system according to the manufacturer’s directions (Amersham, Little Chalfont, UK). Intensities of immunoreactive protein bands were quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). All experiments were repeated a minimum of three times.
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