The level of interleukin-1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), phosphorylated 4E-binding protein 1 (p-4E-BP1), phosphorylated mammalian target of rapamycin (p-mTOR), P70 ribosomal protein S6 kinase 1 (p70S6K1), muscle atrophy F-box (MAFbx), and muscle RING-finger protein-1 (Murf1) proteins in muscle tissue were measured by western blotting. The pooled muscle tissues were homogenated with lysis buffer. Next, the protein concentration of homogenates was quantitated by a Nanodrop spectrophotometer (Maestrogen Inc., Taiwan). An equal amount of protein loaded to sample well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoresis, proteins were transferred into nitrocellulose membranes (0.45 µm); unspecific proteins were blocked by 5% bovine serum albumin. The nitrocellulose membranes were incubated with primary antibodies (IL-1β, IL-6, TNF-α, PGC-1α, p-4E-BP1, p-mTOR, p70S6K1, MAFbx, MuRF-1, Abcam, UK) overnight at 4 °C. β-actin protein was used to control protein loading. Specific binding between primary and secondary antibodies was visualized with diaminobenzidine (DAB) substrate. The protein bands were analyzed densitometrically with the Image J software (National Institute of Health, Bethesda, MD, USA). Blots were performed at least three times.
Murf1
Manufactured by Abcam
Sourced in United States, United Kingdom
MuRF1 is a protein that plays a role in the regulation of muscle mass and function. It belongs to the MuRF family of proteins and is involved in the ubiquitination and degradation of muscle-specific proteins.
Automatically generated - may contain errors
Lab products found in correlation
Atrogin 1, by Abcam (9 mentions)
Mafbx, by Abcam (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Fluorescent secondary antibody, by Cell Signaling Technology (2 mentions)
Smp30, by Abcam (2 mentions)
Hdac2, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
P67phox, by Abcam (2 mentions)
Na k atpase, by Abcam (2 mentions)
Pgc 1α, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Gapdh, by HyTest (2 mentions)
Enhanced chemiluminescence system, by Kodak (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
0.2 m pvdf membrane, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Perilipin, by Abcam (1 mentions)
25 protocols using murf1
1
Muscle Protein Biomarkers Analysis
2
Western Blot Analysis of Muscle Proteins
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C2C12 cells and gastrocnemius muscle were lysed with RIPA buffer (Solarbio, Beijing, China); the protein concentrations were measured by a BCA kit (Beyotime, Shanghai, China). Then, 40 µg of the samples were electrophoresed in 10–12% SDS polyacrylamide gels and electrically transferred to 0.2-µm PVDF membranes (Millipore, USA). The membranes were immersed and incubated in 5% fat-free milk at room temperature for 1 hour. The membranes were then incubated with primary antibodies against HDAC2, P53, P21, IKK, and NF-κBp65 (1:1000, Cell Signaling Technology, USA) and MURF1, MAFbx, and SMP30 (1:1000, Abcam, UK) overnight at 4°C, followed by incubation with fluorescent secondary antibodies (1:1000, Cell Signaling Technology, USA). The GAPDH antibody (1:1000, Cell Signaling Technology, USA) was used as the control. Fluorescence signal detection was performed with an infrared imaging instrument (Odyssey, USA), and protein expression was quantified by densitometry analysis with ImageJ software.
3
Immunohistochemical Analysis of Mouse Subcutaneous Tissues
4
Western Blot Analysis of Protein Signaling
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Protein (50 μg) was separated by 10% and 12% SDS-polyacrylamide gel electrophoresis, based on the molecular weight of the protein of interest and transferred onto a nitrocellulose membrane (Millipore, Billerica, USA). The membranes were blocked with 3% bovine serum albumin in PBS containing 0.1% Tween 20 (Sigma), washed and probed with respective mouse/rabbit monoclonal antibodies. Primary antibodies PDI, Akt, p-Akt and CHOP/GADD153 were obtained from Santa Cruz Biotech, p70S6kinase from Cell Signaling Technology (MA, USA), GRP-78, GSK-3β and NFκB from Sigma (St. Louis, MO, USA) while MAFbx-1 and MuRF-1 from abcam. The membranes were then incubated with anti-mouse/rabbit-IgG HRP conjugate (Sigma). The membrane was washed and incubated with chemiluminescent substrate (Sigma) and the bands were developed using Gel Documentation System (UVP Bioimaging software, Upland CA, USA). Quantification was performed by densitometry using ImageJ software. GAPDH from Sigma (St. Louis, MO, USA) was used as an internal (loading) control.
5
Western Blotting of Lung Proteins
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Equal amounts of mouse lung homogenate were separated by SDS-polyacrylamide gel electrophoresis (PAGE) for Western blotting with specific antibodies against fibronectin (Millipore, Billerica, MA), Atrogin-1, MuRF1 (Abcam, Cambridge, MA, USA), GAPDH, ST2 (GeneTex Inc, Irvine, CA), α-tubulin (Santa Cruz), p-STAT3 (Tyr705), STAT3, p-AMPKα (T172), AMPKα, p-AKT (S742), and AKT (Cell Signaling, Beverly, MA, USA).
6
Protein Expression Analysis via Western Blot
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The nuclear protein extraction kit (Beyotime, China) was used to get total protein of samples. Then, 20μg protein was separated via 12% SDS-PAGE and transferred to PVDF membranes before being blocked with 5% milk for 1 hour at room temperature. The PVDF membranes were stained at 4°C overnight with antibodies specific for FoxO1 (1:1000, Abcam, England), MuRF1(1:1000, Abcam, England), MyoG (1:1000, Abcam, England) and Lamin B1 (1:1000, Abcam, England). Finally, Blots were then stained with fluorescence secondary antibodies (1:20,000, Rockland, USA) and analyzed by the Odyssey Infrared Imaging System (Li-COR Biosciences).
7
Western Blot Analysis of Adipogenic and Myogenic Markers
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Tissue samples were lysed in lysis buffer (iNtRON Biotechnology, Seoul, Korea), and lysate protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by SDS-PAGE gel electrophoresis and transferred to membranes. Membranes were blocked with 5 % skimmed milk and incubated with the indicated primary antibodies overnight at 4 °C. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Antibodies against C/EBPα, PPARγ, FABP4, p-PKA (Ser 114), PGC1α, MyoD, myogenin, and MEF-2 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies against ATGL, p-HSL (Ser 563), and p-AMPK (Thr172) were purchased from Cell Signaling Technology (Danvers, MA, USA); antibodies against MGL, PPARα, PRDM16, UCP1, NRF1, TFAM, UCP3, Atrogin-1, MuRF1, and MYH7 were purchased from Abcam (Cambridge, UK); and an antibody against β-actin was purchased from ABM (Richmond, BC, Canada).
8
Immunoblotting Analysis of Muscle Proteins
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Commercial antibodies used for immunoblotting in this study were S100A8 (Santa Cruz Biotechnology, CA, USA); S100A9 (Genetex, Irvine, CA, USA); Atrogin-1 (Abcam, Cambridge, UK); MuRF1 (Abcam, Cambridge, UK) and GAPDH (Santa Cruz Biotechnology, CA, USA). Protein extracts were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane. For detection of target proteins, the skimmed milk blocked membranes were incubated with specified primary antibodies and corresponding secondary antibodies conjugated with horseradish peroxidase. The bound antibodies were detected using enhanced chemiluminescence reagent (Merck Millipore, Burlington, MA, USA). Quantification was performed by densitometry using ImageQuant version 5.2 software (GE Healthcare, Chicago, IL, USA).
9
Western Blot Analysis of Cardiac Proteins
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40 μg of protein from heart tissue or cell lysates or 10 μg of protein from isolated membrane lysates were loaded onto SDS-polyacrylamide gels. After electrophoresis, the separate proteins were transferred onto Bio-Rad polyvinylidene difluoride membranes. After blocking in 5% nonfat milk for 1 h, the membranes were incubated with antibodies against NOX4 (1:1000 dilution; Abcam), Rac1 (1:1000 dilution; Abcam), MuRF1 and atrogin-1 (1:1000 dilution; Abcam), Na+/K+-ATPase (1:1000 dilution; Abcam), p67phox (1:1000 dilution; Abcam), phosphorylation of Ser-345 and Ser-370 on p47phox and total p47phox (1:1000 dilution; Thermo Fisher Scientific Inc.), phosphorylated p38 and total p38 (1:1000 dilution; Cell Signaling Technology), phosphorylated ERK1/2 and total ERK1/2 (1:1000 dilution; Cell Signaling Technology), and GAPDH (1:5000 dilution; Cell Signaling Technology), respectively, followed by secondary relevant antibodies conjugated with horseradish peroxidase. The signals were then developed using an enhanced version of the chemiluminescence reaction.
The protein ladders were purchased from FroggaBio Inc. (Concord, Canada) for cultured cardiomyocytes (245, 180, 135, 100, 75, 63, 48, 35, 25, 20, 17, and 11 kDa) and Thermo Fisher Scientific China Co. Ltd. for heart tissue samples (170, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kDa).
The protein ladders were purchased from FroggaBio Inc. (Concord, Canada) for cultured cardiomyocytes (245, 180, 135, 100, 75, 63, 48, 35, 25, 20, 17, and 11 kDa) and Thermo Fisher Scientific China Co. Ltd. for heart tissue samples (170, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kDa).
10
Western Blot Protein Detection
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The cell lysates were prepared in lysis buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton X-100) at 4 °C. The extractions were separated via SDS-PAGE, transferred onto a polyvinylidine difluoride membrane (Millipore, Bedford, MA, USA), and detected using antibodies against H3, p-NFκB.p65 (S536) (Cell Signaling Technology, Danvers, MA, USA), α-actinin (ACTN), p53, p21, Cyclin D1, H3P, MYH, Twist, COX-2, ATF-3, NFκB-p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), γH2A.X, myogenin, MuRF1, and Fbx32 (Abcam, Cambridge, UK). The membranes were incubated first with primary antibodies against proteins of interest and then with HRP-conjugated secondary antibodies (anti-mouse IgG, AP192P and anti-rabbit IgG, AP132P, Merck-Millipore, Burlington, MA, USA). The immunoreactive proteins were detected using ECLTM Western Blotting Detection Reagent and Amersham HyperfilmTM ECL (GE Healthcare, Waukesha, WI, USA). The procedural details have been described in our previous publications [47 (link),48 (link)].
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