Diaphragm muscles were isolated from 2-, 4-, and 24-week-old mdx and mdx/IL6 mice and immediately frozen in liquid nitrogen. Each sample (liquid nitrogen powdered diaphragm muscles) was homogenized in protein lysis buffer [Tris-HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, cocktail protease inhibitors/1X (Sigma Aldrich)]. Western blotting analysis was performed using 70 μg of protein extracts, and filters were blotted with antibodies against gp91phox (BD Transduction Laboratories), Nrf2 (Santa Cruz Biotechnology), NFκB p65 (ser536; Cell Signaling), NFκB (Cell Signaling), α-tubulin (Sigma Aldrich), β-tubulin (Cell Signaling), Glu-tubulin (detyrosinated α-tubulin; Abcam), and GAPDH (Santa Cruz Biotechnology). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories), and densitometric analysis was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories©).
Chemi doctm xrs 2015
Manufactured by Bio-Rad
Sourced in United States
The Chemi DocTM XRS 2015 is a gel imaging system designed for the detection and analysis of chemiluminescent and fluorescent signals in biological samples. The system is equipped with a high-resolution CCD camera, allowing for the capture of high-quality images. It is capable of imaging a variety of sample types, including Western blots, nucleic acid gels, and protein gels.
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Lab products found in correlation
Image lab software, by Bio-Rad (7 mentions)
Hrp conjugated secondary antibody, by Fortis Life Sciences (3 mentions)
Frataxin, by Santa Cruz Biotechnology (3 mentions)
Lite ablot extend long lasting chemiluminescent substrate, by Euroclone (3 mentions)
Gapdh, by Merck Group (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Gp91phox, by BD (1 mentions)
α tubulin, by Merck Group (1 mentions)
Cocktail protease inhibitors 1x, by Merck Group (1 mentions)
Glu tubulin, by Abcam (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor mini tablet, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Vinculin, by Merck Group (1 mentions)
Hsp60, by Merck Group (1 mentions)
Sc 2005, by Santa Cruz Biotechnology (1 mentions)
Sc 2305, by Santa Cruz Biotechnology (1 mentions)
T5168, by Merck Group (1 mentions)
7 protocols using chemi doctm xrs 2015
1
Molecular Profiling of Dystrophic Diaphragm Muscles
2
Protein Expression Analysis by Western Blot
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Cells were homogenized and lysed on ice with RIPA buffer, including DTT and protease inhibitors. For Western blotting analysis, 40 μg of proteins was subjected to SDS PAGE on 4–12% denaturing gel and probed with the following antibodies: NRF2 (1:500, Abcam, Cambridge, UK), NQO1 (1:1000, Novus Biologicals, Minneapolis, MN, USA), SOD 1/2 (1:5000, Stressgen, Victoria, BC, Canada), Frataxin (1:500, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (1:15,000, Sigma Aldrich, St. Louis, MO, USA) as loading control. Immunoreactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent substrate (Euroclone, MI, Italy). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analysis was performed using Image Lab software (Version 5.2.1; © Bio-Rad Laboratories).
3
Western Blot Analysis of Nrf2 and Nox2
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Leukocytes were lysed on ice with RIPA buffer (Sigma Aldrich, St. Louis, Missouri, USA), including DTT and protease inhibitors (Protease and Phosphatase Inhibitor Mini Tablets, Thermo Scientific, Waltham, MA, USA). Proteins, in the amount of 40 μg, were subjected to SDS PAGE on 4–12% denaturing gel and probed with the following antibodies: Nrf2 (1:500, Abcam, Cambridge, UK), Nox2 (1:2000, Abcam, Cambridge, UK), and GAPDH (1:10,000, Sigma Aldrich) as loading control. Immuno-reactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent kit (Euroclone, Milan, Italy). HRP-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) signals were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry was performed by Image Lab software (Version 5.2.1, Bio-Rad Laboratories). Proteins were quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).
4
Protein Expression Analysis of Fibroblasts
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Fibroblasts (1 × 106) were lysed on ice with RIPA buffer, including DTT and protease inhibitors. 40 µg proteins were subjected to SDS PAGE on 4–12% denaturing gel and probed with the following antibodies: Nrf2 (1:500, Abcam, Cambridge, UK), Nqo1 (1:7000, Novus Biologicals, Minneapolis, MN, USA), Ho-1 (1:3000, Abcam), Frataxin (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:10,000, Sigma Aldrich) as loading control. Immunoreactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent substrate (Euroclone, Milan, Italy). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analysis was performed using Image Lab software (Version 5.2.1, Bio-Rad Laboratories)
5
Immunoblotting of Mouse Heart Proteins
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Mouse hearts were homogenized (1:10 w/v) in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40, and protease and phosphatase inhibitors). Fibroblasts (1 × 106) were lysed on ice with RIPA buffer, including DTT and protease inhibitors. Fourty μg proteins were subjected to SDS PAGE on 4–12% denaturing gel and probed with the following antibodies: NRF2 (1:1000, Thermo Fisher Scientific, USA), GPX4 (1:1000, Thermo Fisher Scientific, USA), Frataxin (1:500, Santa Cruz Biotechnology, USA), Hsp60 (1:10000) and vinculin (1:10000, Sigma) as loading controls. Immunoreactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent substrate (Euroclone, Milan, Italy). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analysis was performed using Image Lab software (Version 5.2.1, Bio-Rad Laboratories).
6
Nuclear Nrf2 and System Xc- Expression Analysis
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To measure Nrf2 nuclear levels, equal amounts of nuclear extracts (20 μg proteins/sample) were loaded in an 8% polyacrylamide gel, subjected to electrophoresis and transferred to nitrocellulose. After incubation with 5% non-fat dry milk for 1 h, membranes were incubated at 4 °C overnight with the polyclonal anti-Nrf2 antibody (1:1000) or with a polyclonal anti-lamin A (1:1000). To analyze System Xc− expression, equal amounts of total extracts (15 μg proteins/sample) were subjected to SDS-PAGE. Electrophoresis was performed using a 10% polyacrylamide gel. Membranes were blotted with anti-xCT polyclonal antibody (1:5000) or polyclonal anti-actin antibody (1:1000).
Actin and lamin A were used as reference proteins for total and nuclear extracts, respectively. Anti-rabbit secondary antibody labeled with peroxidase was used at 1:10,000 dilution. ECL Western blotting detection reagents was used to detect immunoreactive bands that were captured by Chemi Doc TM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA). Densitometric analysis was carried out using Image Lab software (Version 5.2.1; © Bio-Rad Laboratories).
Actin and lamin A were used as reference proteins for total and nuclear extracts, respectively. Anti-rabbit secondary antibody labeled with peroxidase was used at 1:10,000 dilution. ECL Western blotting detection reagents was used to detect immunoreactive bands that were captured by Chemi Doc TM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA). Densitometric analysis was carried out using Image Lab software (Version 5.2.1; © Bio-Rad Laboratories).
7
Western Blot Analysis of Protein Expression
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After treatments, the cells were lysed in 150 μl of lysis buffer 1X (CST) containing 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentration was measured by BCA protein assay kit (Pierce Biotechnology). Samples containing equal amounts of proteins (50 μg) were subjected to SDS-PAGE on a 10% or 15% acrylamide gel 22 (link) and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Whatman GmbH). The filters were blocked for 2 h in Tris-buffered saline-Tween 20 (TBST) and then incubated overnight at 4°C with the primary antibodies. The mouse anti-Muscarinic Acetylcholine Receptor M2/CHRM2 monoclonal IgG1 (1:800, NB120-2805; Novus Bio), rabbit anticleaved caspase 3 (1:500, #9661; CST) were used. Glyceraldehyde-3-phosphate dehydrogenase (1:500, sc-47724; Santa Cruz Biotechnology) and tubulin (1:500, T5168; Sigma-Aldrich) were used to normalize the intensity of the bands. Membranes were incubated with secondary HRP antibodies (anti-mouse, sc-2005, anti-rabbit, sc-2305, 1:10000; Santa Cruz Biotechnology) for 1 h at RT. Signals were detected by an ECL immunodetection system (Amersham Corp.) following the manufacturer's instruction and visualized by Chemi DocTM XRS 2015 (Bio-Rad Laboratories). Densitometric analysis of the bands was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories).
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