Murf1

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany

MuRF1 is a protein that plays a key role in the regulation of muscle protein degradation and atrophy. It functions as an E3 ubiquitin ligase, targeting specific proteins for proteasomal degradation. MuRF1 is involved in the ubiquitination and breakdown of contractile and structural proteins in skeletal muscle.

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Anti-MuRF1 (7 citations) Mouse anti-MURF1 (3 citations) Goat polyclonal antisera to MuRF1 (2 citations)

37 protocols using murf1

Western Blot Analysis of Tissue Proteins

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Western blot analysis was performed with the same method used in a previous study [25 (link)]. Cytosolic and nuclear proteins were extracted from the liver, gastrocnemius tissue, and hippocampus with lysis buffers containing protease/phosphatase inhibitor, cytosol-extracting buffer. A total of 30 μg of protein for each group was used for western blot analysis. The extract was separated by 8~12% SDS-PAGE gel electrophoresis then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Then, it was incubated overnight with primary antibodies and probed with the respective secondary antibody reagent (Biorad, Hercules, CA, USA). The bands were adjusted by the band levels of α-tubulin (cytosol) or PCNA (nucleus) [25 (link)].
Antibody list: pAkt (sc-81434), Akt (sc-514032), GLUT4 (sc-53566), GLUT2 (sc-518022), FGF21 (sc-81946), KYN (sc-69890), PPARα (sc-398394), FGFR1 (sc-57132), CTSB (sc-365558), PGC1α (sc-517380), Foxo3a (sc-48348), Atroin-1 (sc166806), Murf-1 (sc-398608) (Santa Cruz Biotechnology, CA, USA, 1:200), IRS-1 (#2382), pIRS-1 (#2381), mTOR (#2972), pmTOR (#2971), LC3 (#2775), (#2535), AMPK (#2532) (cell signaling technology, MA, USA, 1:2000), FNDC5 (ab131390), BDNF (ab108319), β-klotho (ab127879) (Abcam, Cambridge, MA, USA, 1:10,000), α-tubulin (T5168, Signa Aldrich, MO, USA, 1:4000), PCNA (610665, Enzolife science, Farmingdale, NY, USA, 1:1000).

Lee H., Kim S.Y, & Lim Y. (2023). Annona muricate Extract Supplementation Contributes to Improve Aberrant Multi-Organ Energy Metabolism via Muscle–Brain Connectivity in Diabetic Mice. Nutrients, 15(11), 2559.

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Muscle Protein Regulation Analysis

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Expression of Atrogin, MuRF1, MyHC, STAT3, and pSTAT3 was determined using western blot analysis. Atrogin (mouse monoclonal IgG1, 1:500) and MuRF1 (mouse monoclonal IgG1, 1:1000) antibodies were obtained from Santa Cruz, CA, USA. STAT3 (rabbit monoclonal IgG, 1:1000) and pSTAT3 (rabbit monoclonal IgG, 1:1000) antibodies were purchased from Cell Signaling Technology (Danvers, MA). MyHC (MF-20, mouse monoclonal IgG, 1:000) was obtained from Developmental Studies Hybridoma Bank, University of Iowa, USA. The anti-nitrotyrosine antibody (ab61392, mouse monoclonal IgG2a, 1:100 dilution) was purchased from Abcam (Cambridge, UK).

Shukla S.K., Markov S.D., Attri K.S., Vernucci E., King R.J., Dasgupta A., Grandgenett P.M., Hollingsworth M.A., Singh P.K., Yu F, & Mehla K. (2020). Macrophages Potentiate STAT3 Signaling in Skeletal Muscles and Regulate Pancreatic Cancer Cachexia. Cancer letters, 484, 29-39.

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Protein Extraction and Western Blotting for Cell and Tissue Analysis

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Total proteins of C2C12, 3T3-L1 cells, muscle, or fat tissues were extracted using RIPA buffer supplemented with protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), and protein concentrations were measured using the Bradford assay. For western blotting, proteins (40 or 60 µg) were electrophoresed in 6, 10, or 12% SDS-polyacrylamide gels and transferred to PVDF membranes (EMS-Millipore, Billerica, MA, USA). Membranes were then incubated in blocking reagent (3% skim milk or BSA in Tris-buffered saline (TBS)/Tween 20) and treated overnight with specific primary antibodies (FMOD (1:400), MSTN (1:1000), Pax7 (1:500), MYOD (1:500), MYOG (1:500), MYH (1:500), MuRF1 (1:500), Atrogin1 (1:500), or β-actin (1:1000) antibodies (Santa Cruz Biotechnology), or MYL2 (1:1000, Abcam, Cambridge, MA, USA), ACVRIIB (1:500, Abcam)) in 1% skim milk or BSA in TBS at 4 °C. After washing, membranes were incubated with goat-rabbit or mouse-horseradish-peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Blots were developed using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Lee E.J., Shaikh S., Baig M.H., Park S.Y., Lim J.H., Ahmad S.S., Ali S., Ahmad K, & Choi I. (2022). MIF1 and MIF2 Myostatin Peptide Inhibitors as Potent Muscle Mass Regulators. International Journal of Molecular Sciences, 23(8), 4222.

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Protein expression analysis in cell extracts

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Cells were harvested and homogenized in RIPA buffer (Sigma-Aldrich^® GmbH, St. Louis, MO, USA) containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.1% (v/v) sodium dodecyl sulfate and protease, phosphatase inhibitor (Roche, Basel, Switzerland). Protein concentrations were determined by DC™ Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and equal amounts of proteins (25 µg) were separated by 8% and 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with antibodies specific to Myosin (1:200), MuRF-1 (1:200), and Foxo (1:200) (all from Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:10,000, Acris Antibodies GmbH, Herford, Germany), respectively, and appropriate secondary dye-conjugated antibodies goat anti-mouse IRdye800, IRdye650 (1:10,000; LI-COR^®, Lincoln, NE, USA) to reveal protein bands for 1 h at room temperature. Membranes were scanned using the Odyssey SA Imaging System (LI-COR^®, Lincoln, NE, USA). Experiments were performed in duplicates and the signal intensity was normalized to GAPDH.

Langendorf E.K., Rommens P.M., Drees P., Mattyasovszky S.G, & Ritz U. (2020). Detecting the Effects of the Glucocorticoid Dexamethasone on Primary Human Skeletal Muscle Cells—Differences to the Murine Cell Line. International Journal of Molecular Sciences, 21(7), 2497.

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Protein Isolation and Western Blotting

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Protein isolation and Western blotting were performed as described previously (Shukla et al., 2015 (link)). Briefly, cells were washed twice with PBS and lysed in radioimmunoprecipitation assay lysis buffer by shaking on ice for 10 min. Then, the lysates were centrifuged at 13,000 rpm for 5 min, and the supernatant was collected. Protein concentration was measured by the Bradford assay. Equal amounts of protein were loaded into the SDS-PAGE gel for immunoblotting. Primary antibodies against MuRF1, ATROGIN-1, and p65 (NF-κB; Santa Cruz Biotechnology); SIRT1, FOXO3a, FOXO1, and Acetyl-p65 (K310; Cell Signaling Technology); tubulin, actin (JLA20), and MyHC (Developmental Studies Hybridoma Bank); and NOX4 (Abcam) were used for probing specific proteins. Loading control was run in parallel on separate gels for each experiment.

Dasgupta A., Shukla S.K., Vernucci E., King R.J., Abrego J., Mulder S.E., Mullen N.J., Graves G., Buettner K., Thakur R., Murthy D., Attri K.S., Wang D., Chaika N.V., Pacheco C.G., Rai I., Engle D.D., Grandgenett P.M., Punsoni M., Reames B.N., Teoh-Fitzgerald M., Oberley-Deegan R., Yu F., Klute K.A., Hollingsworth M.A., Zimmerman M.C., Mehla K., Sadoshima J., Tuveson D.A, & Singh P.K. (2020). SIRT1–NOX4 signaling axis regulates cancer cachexia. The Journal of Experimental Medicine, 217(7), e20190745.

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Western Blot Analysis of Muscle Signaling

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Total cell lysates were solubilized in ice-cold RIPA lysis buffer (Beyotime Biotechnology, P0013B, China) consisting of protease and phosphatase inhibitor cocktails (HY-K0010, HY-K0021, HY-K0022, MCE, United States). The membranes were blocked with 5% bovine albumin for 2 h at room temperature prior to incubation with the indicated primary antibodies. The signals were detected with the following antibodies following standard procedures: phosphorylated STAT3 (CST, #9139), Atrogin-1 (Satan Cruz, sc166806), MuRF-1 (Santa Cruz, sc398608), MHC (R&D Systems, #MAB4470), p62 (CST, #5114s), LC3 (Proteintech, 14600-1-AP), phospho-P70 S6K (Beyotime, AF5899), phospho-EIF4EBP1 (Beyotime, AF5806), phospho-mTOR (Beyotime, AF5869), GDF8/Myostatin (Proteintech, 19142-1-AP), iNOS (Affinity, AF0199), phospho-Akt (Ser473) (CST, #4060), phospho-AMPK (Abcam, ab133448), ATGL (CST, #2439), HSL (CST, #18381), UCP-1 (Satan Cruz, sc518171) and GAPDH (Proteintech, 60004-1-Ig). Subsequently, the membranes were washed and incubated for 2 h at room temperature with peroxidase-conjugated secondary antibodies (Bioworld). Following several washes, chemiluminescent images of immunostained bands on the membranes were recorded on X-ray films using the enhanced chemiluminescence (ECL, FUDE, FD8020, China) system according to the manufacturer’s instructions.

Wei L., Wang R., Lin K., Jin X., Li L., Wazir J., Pu W., Lian P., Lu R., Song S., Zhao Q., Li J, & Wang H. (2022). Creatine modulates cellular energy metabolism and protects against cancer cachexia-associated muscle wasting. Frontiers in Pharmacology, 13, 1086662.

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Muscle Protein Expression Analysis

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Excised muscles were homogenized with a pestle and lysed for protein in RIPA Lysis and Extraction buffer (Fisher Scientific) with EDTA‐free protease inhibitor cocktail (Sigma‐Aldrich). All muscles were lysed individually. Likewise, differentiated C2C12 myotubes were lysed following a 24 h incubation with the indicated conditioned media. Western blots were run using antibodies for tryptase (Abcam, cat #: ab151757), Mas‐related gene X2 (MrgX2) (1:1000, Novus Biological, Littleton, CO, cat #: NB110‐75035), CD68 (1:1,000, Abcam, cat #: ab125212), myeloperoxidase (MPO) (1:1000, Abcam, cat #: ab208670), muscle‐specific RING finger‐1 (MuRF1) (1:1000, Santa Cruz Biotechnology, Dallas, TX, cat #: sc‐398608), muscle atrophy F‐box (MAFbx) (1:1000, Santa Cruz Biotechnology, cat #: sc‐166806), and fast myosin skeletal heavy chain (MyHC) (1:1,000, Abcam, cat #: ab91506). GAPDH (1:5000, Cell Signalling Technology, Danvers, MA, cat #: 2118) was used as the loading control. Expression of target proteins was normalized based on GAPDH expression using ImageJ.

Widner D.B., Liu C., Zhao Q., Sharp S., Eber M.R., Park S.H., Files D.C, & Shiozawa Y. (2021). Activated mast cells in skeletal muscle can be a potential mediator for cancer‐associated cachexia. Journal of Cachexia, Sarcopenia and Muscle, 12(4), 1079-1097.

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Cellular Signaling Pathway Analysis in Frozen Muscle

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Frozen gastrocnemius muscle tissue was ground into small pieces in liquid nitrogen and lysed in a lysis buffer containing cOmplete™ Protease Inhibitor Cocktail and PhosSTOP™ (Roche Diagnostics, Indianapolis, IN, USA), and then centrifuged. A Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to measure the protein concentration and same concentration of protein of each group was subjected to SDS-PAGE. After transferring, membranes were blocked by 5% skim milk and incubated with a primary antibody overnight. The primary antibodies used for Western blot were p-Akt, Akt, p-mTORc1, mTORc1, p-S6K1, S6K1, p-4E-BP1, 4E-BP1, p-FoxO3a, and FoxO3a (Cell signaling, MA, USA), MuRF1 and Atrogin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (GeneTex, Irvine, CA, USA). The next day, the membranes were incubated with the corresponding secondary antibodies for visualizing the protein bands using LAS3000 luminescent image analyzer (Fuji Film, Tokyo, Japan). β-actin was used as a loading control and Image J software (National Institute of Health, Bethesda, MD, USA) was used for quantification.

Han M.J., Park S.J., Lee S.J, & Choung S.Y. (2022). The Panax ginseng Berry Extract and Soluble Whey Protein Hydrolysate Mixture Ameliorates Sarcopenia-Related Muscular Deterioration in Aged Mice. Nutrients, 14(4), 799.

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Skeletal Muscle and Adipose Tissue Protein Extraction

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The protein extraction of skeletal muscle (gastrocnemius) adipose tissue (epididymis fat) was performed according to a method previously described [36 (link),37 (link)]. Protein was quantified using a BCA Protein Assay Kit (Abcam, Cambridge, MA, USA). The following primary antibodies are used: PGC-1α, pAMPK, AMPK, FoxO3a, Atrogin-1, MuRF1, Bax, Bcl-xL, Akt, pAkt (Santa Cruz Biotechnology, CA, USA, 1:200); S6K1 (Cell Signaling Technology, MA, USA, 1:1000); α-tubulin (Sigma-Aldrich, St. Louis, MO, USA, 1:4000); IGF-1 (Abcam, Cambridge, MA, USA, 1:1000); β-Actin (Bethyl Laboratories, Montgomery, TX, USA, 1:500); PCNA (Enzolife science, Farmingdale, NY, USA, 1:1000).

Lim G, & Lim Y. (2022). Effects of Whey Peptide Supplementation on Sarcopenic Obesity in High-Fat Diet-Fed Mice. Nutrients, 14(20), 4402.

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Protein Extraction and Western Blot Analysis

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Total cell lysates were solubilized in ice-cold RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor cocktails (MedChemExpress (Monmouth Junction, NJ, USA). Incubation with the primary antibodies was performed after blocking the membranes with 5% bovine albumin at room temperature for 2 h. Standard procedures were followed for the detection of the signals using the following antibodies: STAT3 (CST, #9145), phosphorylated STAT3 (CST, #9139), atrogin-1 (Satan Cruz, sc166806), MuRF-1 (Santa Cruz, sc398608), MHC (R&D Systems, #MAB4470), p62 (CST, #5114s), LC3 (Proteintech, 14600-1-AP), cleaved caspase3 (CST, #9664T), cleaved PARP (CST, #5625), phospho-P70 S6K (Beyotime, AF5899), phospho-EIF4EBP1 (Beyotime, AF5806), GLUT1/SLC2A1 rabbit mAb (Abclonal, A11727), Bdh1 rabbit pAb (Abclonal, A3763), HMGCS2 antibody (Affinity, DF14319), and GAPDH (Proteintech, 60004-1-Ig). Subsequently, at room temperature, the membranes were washed and incubated for 2 h with peroxidase-conjugated secondary antibodies (Bioworld). The enhanced chemiluminescence (ECL) system was used to record chemiluminescent images of immunostained bands on membranes on X-ray films, following the manufacturer’s protocol.

Wei L., Wang R., Wazir J., Lin K., Song S., Li L., Pu W., Zhao C., Wang Y., Su Z, & Wang H. (2022). 2-Deoxy-D-glucose Alleviates Cancer Cachexia-Induced Muscle Wasting by Enhancing Ketone Metabolism and Inhibiting the Cori Cycle. Cells, 11(19), 2987.

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