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©).
Gp91phox
Manufactured by BD
Sourced in United States
Gp91phox is a subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. It plays a core role in the production of superoxide, a reactive oxygen species, in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
P47phox, by Merck Group (4 mentions)
Cocktail protease inhibitors 1x, by Merck Group (3 mentions)
P47phox, by Santa Cruz Biotechnology (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Hrp conjugated secondary antibody, by Fortis Life Sciences (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Image acquisition and analysis software, by Bio-Rad (2 mentions)
P22phox, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Abcam (2 mentions)
P47phox, by BD (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
α tubulin, by Merck Group (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Glu tubulin, by Abcam (1 mentions)
Chemi doctm xrs 2015, by Bio-Rad (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Collagen 1, by Abcam (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Ferritin heavy chain, by Abcam (1 mentions)
18 protocols using gp91phox
1
Molecular Profiling of Dystrophic Diaphragm Muscles
2
Renal Injury Protein Profiling
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Mouse renal tissues were collected and homogenized in lysis buffer. The homogenates were centrifuged at 16,000×g at 4°C for 15 min. A bicinchoninic acid protein assay kit (Pierce Co, Rockford, IL, USA) was used to analyze the protein concentrations. Equal amounts (20 µg) of the protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis geland transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and incubated with the following primary antibodies overnight: cleaved caspase-3 (1:1000; Abcam, Cambridge, MA, USA), KIM-1 (1:1,000; Abcam, Cambridge, MA, USA), NGAL (1:1,000; Abcam, Cambridge, MA, USA), collagen I (1:1,000; Abcam, Cambridge, MA, USA), HIF-1α (1:1,000; Novus Bio, Littleton, CO, USA), HO-1 (1:1,000; BD transduction, San Jose, CA, USA), ferritin heavy chain (1:1000; Abcam, Cambridge, MA, USA), gp91 phox (1:1,000; BD transduction, San Jose, CA, USA), GPX4 (1:1,000; Abcam, Cambridge, MA, USA), and β-actin (1:1,000; Cell Signaling, Danvers, MA, USA). After washed, the membranes were incubated for 2 h with a secondary antibody coupled to horseradish peroxidase (1:5,000; Santa Cruz, CA, USA). Densitometric analyses were carried out with image acquisition and analysis software (Bio-Rad).
3
Protein Extraction and Antibody Detection in Myocardial Tissues
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The preparation of protein samples from myocardial tissues was performed as previously described (Becker et al. 2005 (link)). Antibodies to eNOS (BD Transduction Laboratories, 1:250 dilution), phospho-eNOS (Ser 1177) (Cell Signal, 1:500 dilution), SOD-1(Calbiochem, 1:5000 dilution), SOD-2 (BD Transduction Laboratories, 1:10,000 dilution), SOD-3 (Santa Cruz Biotechnology, 1:5000 dilution), or one of the following subunits of NAD(P)H oxidase: p67phox (Upstate, 1:1000 dilution), p22phox (Santa Cruz Biotechnology, 1:2000 dilution), gp91phox(BD Transduction Laboratories, 1:1000 dilution), p47phox (Santa Cruz Biotechnology, 1:1000 dilution), phos-p47phox (Upstate, 1:1000 dilution), p40phox (Santa Cruz Biotechnology, 1:800 dilution), Rac-1 (Santa Cruz Biotechnology, 1:5000 dilution), and nitrotyrosine (Santa Cruz Biotechnology, 1:1000 dilution) were used.
4
Western Blot Analysis of Oxidative Stress Markers
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Total cytosolic and plasma membrane proteins were prepared as described previously27 (link). Equal amounts of protein were separated by 4–12% b-tris-polyacrylamide electrophoresis gel and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibody against 4-hydroxy-2-nonenal (4-HNE), αM, phospho-ERK1/2, ERK1/2, phospho-Src, Src (Cell Signaling Technology, Danvers, MA, USA), p47phox (1:1,000; EMD Millipore, Temecula, CA, USA), gp91phox (1:1,000; BD Transduction Laboratories, San Jose, CA, USA), and GAPDH (1:1000, Abcam, Cambridge, MA, USA) overnight at 4 °C and followed by horseradish peroxidase-linked anti-rabbit IgG (1:3000) for 2 h at 25 °C. ECL reagents (Biological Industries, Cromwell, CT, USA) were used as a detection system.
5
Paraquat-Induced Neuroinflammation Model
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Paraquat, maneb and taurine were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). RNAiso Plus and SYBR Premix Ex TaqTM II were obtained from Takara Bio Inc. (Takara, Tokyo, Japan). The membrane protein extraction kit was obtained from Beyotime (Jiangsu, China). The following primary antibodies were used: TH (EMD Millipore Corporation, Billerica, MA, USA), α-synuclein (Abcam, Cambridge, MA, USA), Iba-1 (Wako Chemicals, Richmond, VA, USA), CD11b (AbD Serotec, Raleigh, NC, USA), phosphorylated p65, p65, phosphorylated IκBα, IκBα, phosphorylated IKKα (Cell Signaling Technology, Danvers, MA, USA), 4-HNE (Abcam, Cambridge, MA, USA), p47phox (EMD Millipore, Temecula, CA, USA), gp91phox (BD Transduction Laboratories, San Jose, CA, USA) and GAPDH (Abcam, Cambridge, MA, USA). The BCA Protein Assay Kit was purchased from Life Technologies (Waltham, MA USA). All other chemicals were of the highest grade commercially available.
6
Atrial Tissue Protein Analysis
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Right atrial (RA) and left atrial (LA) tissue homogenates were probed with the following antibodies: gp91phox (BD Transduction); total and phosphorylated RyR2 at serine-2808 and serine-2814 (ThermoFisher Scientific); α-smooth muscle actin (α-SMA; Cell Signalling); vimentin (Exbio); HRP-conjugated GAPDH (Sigma Aldrich); and total CaMKII (Cell Signalling) and an antiserum to oxidized CaMKII that was kindly provided by Dr ME Anderson, Johns Hopkins University, USA. Detection of primary antibodies was performed using peroxidase-conjugated anti-rabbit or anti-mouse antibodies (both Promega, USA). GAPDH was used as a loading control except for phosphorylated protein levels which were normalized by the respective total protein levels. All western blots were run under reducing conditions apart from those to detect oxidized CaMKII, which was performed under non-reducing conditions to preserve the redox modification. For the detection of RA and LA NOX2, atrial tissues were pooled from two different animals prior to homogenization.
7
Immunoblotting of Inflammatory Markers
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For western blot analysis, equal amounts of protein were separated by 4−12 % Bis-Tris Nu-PAGE gel and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with 5 % nonfat milk and incubated with rabbit antibodies (1:1000) against Iba-1, p47phox, gp91phox (BD Transduction Laboratories), GAPDH, or rat CD11b antibody overnight at 4 °C. The next day, membranes were incubated with HRP-linked secondary anti-rabbit or rat IgG (1:3000) for 2 h at room temperature. ECL reagents (Amersham Biosciences) were used as a detection system.
8
Western Blot Analysis of Protein Expression
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Homogenate aliquots (50 μg of total protein) were boiled and then electrophoresed in 12% SDS‐PAGE acrylamide gels, transferred onto PVDF membrane (Millipore, Billerica, MA, USA), and incubated for 1 h in TBS‐T (Tris‐buffered saline and 0.1% Tween‐20) containing 5% nonfat milk. Membranes were then incubated with primary antibodies against p‐AMPK (1:1000; Cell Signaling Technology, Danvers, MA, USA), AMPK (1:1000; Cell Signaling Technology), gp91phox (1:1000; BD Transduction Laboratories, Lexington, KY, USA), occludin (1:300; Invitrogen), or claudin‐5 (1:500; Invitrogen) overnight at 4°C, washed in TBS‐T, and then incubated for 1 h at room temperature with corresponding HRP‐conjugated anti‐rabbit or anti‐mouse antibodies (1:2000; Boster). The membranes were developed with the SuperSignal West Pico HRP substrate kit (Pierce, Rockford, IL, USA) and photographed. Protein band intensities were quantitated after normalization to beta actin.
9
Protein Expression Analysis in FDB Muscles
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FDB muscles were homogenized using an electric homogenizer (Fluko, Shanghai, China) in a lysis buffer containing in mM: 20Tris-HCl (pH 7.5), 1% Triton X-100, 2 EDTA, 20 NaF, 1 Na2P2O7, 10% glycerol, 150 NaCl, 10 Na3VO4, 1 PMSF and protease inhibitors (Complete™, Roche Applied Science). Proteins were separated using SDS-PAGE and transferred to PVDF membranes. The following primary antibodies and their dilutions were used as follows: Total NF-κB total p65 subunit (1:1000; Cell Signaling); p-Ser536-p65 (1:1000; Cell Signaling); p47phox (1:5000; Sigma); p-p47phox (pSer359) (1:5000; Sigma); gp91phox (1:2000; BD Transduction); p-p38 (thr180/tyr182) (1:2000; Santa Cruz); p38 (1:2000; Santa Cruz); Phospho-ERK1/2(Thr202/Tyr204)(1:2000; Cell Signaling); ERK 1/2 (1:2000; Santa Cruz); α-Tubulin (1:5000; Cell Signaling); anti-mouse IgG-HRP (1:20,000; Santa Cruz); anti-rabbit IgG-HRP (1:30,000; Thermo Scientific Pierce). The protein bands in the blots were visualized using a WESTAR Supernova detection kit (Cyanagen, Bologna, Italy) and ChemiDoc™ MP System (Bio-Rad, USA). The intensity of the bands was determined with ImageJ densitometry analysis.
10
Protein Expression Analysis in Mouse Left Ventricle
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Mouse left ventricle heart tissues were collected and homogenized in lysis buffer. The homogenates were centrifuged at 16 000 g at 4°C for 15 minutes. The BCA protein assay kit (Pierce Co) was used to analyse the protein concentrations. Equal amounts (20 µg) of the protein were separated by 10% SDS‐PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% non‐fat dry milk in Tris‐buffered saline and incubated with the following primary antibodies overnight: SIRT3 (1:1000; Cell Signaling), β‐MHC (1:1000; Abcam), TGF‐β1 (1:1000), gp91phox and p47phox (1:1000; BD transduction). After washing, the membranes were incubated for 2 hours with an anti‐rabbit or anti‐mouse secondary antibody coupled to horseradish peroxidase (1:5000; Santa Cruz). Densitometric analyses were carried out with image acquisition and analysis software (Bio‐Rad).
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