Murf1

Manufactured by R&D Systems

Sourced in United States

MuRF1 is a protein that functions as an E3 ubiquitin-protein ligase. It plays a role in the ubiquitination and subsequent proteasomal degradation of target proteins.

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Lab products found in correlation

Acss2, by Santa Cruz Biotechnology (1 mentions) Odyssey imager, by LI COR (1 mentions) Goat anti rabbit igg p0448, by Agilent Technologies (1 mentions) Pfoxo1 s256, by Cell Signaling Technology (1 mentions) Ac foxo1, by Santa Cruz Biotechnology (1 mentions) Total foxo1, by Cell Signaling Technology (1 mentions) Amersham hybond p, by GE Healthcare (1 mentions) Nupage gel, by Thermo Fisher Scientific (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Pa1 211a, by Thermo Fisher Scientific (1 mentions) Glycerol 2 phosphate, by Merck Group (1 mentions) Na931v, by GE Healthcare (1 mentions) Anti gst, by Merck Group (1 mentions) Gapdh, by Merck Group (1 mentions) Anti flag, by Merck Group (1 mentions) Phosphatase inhibitor, by Roche (1 mentions)

3 protocols using murf1

Western Blotting of Muscle Atrophy Markers

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Western blot was performed as previously described.^{17 (link)} Cell lysates were loaded into sodium dodecyl sulfate–polyacrylamide gel for electrophoresis. After the electrophoresis, the protein was transferred onto nitrocellulose membrane and blocked in 5% skim milk at room temperature for 1 hour, followed by the incubation with desirable antibodies against ZIP4 (Proteintech, 1:2000), ACSS2 (Santa Cruz Biotechnology, 1:1000), ACTB (Proteintech, 1:10000), MuRF1 (R&D, 1:500), Atrogin-1 (ECM Biosciences, 1:1000), MHC (University of Iowa, 1:1000), SDC1 (Proteintech, 1:1000), p- GSK3β (CST, 1:1000), total- GSK3β (CST, 1:1000), p-CREB (CST, 1:1000), total-CREB (CST, 1:1000), ETV4 (Aviva Systems Biology, 1:1000), or mouse ZIP4 (R&D, 1:1000) at 4°C overnight. Then, the membrane was washed with Tris-Buffered Saline buffer with 0.1% Tween 20, followed by the incubation of IRDye secondary antibodies (1:10,000) at room temperature for 2 hours. The results were examined by Odyssey Imager (LI-COR).

Zhou Z., Ren Y., Yang J., Liu M., Shi X., Luo W., Fung K.M., Xu C., Bronze M.S., Zhang Y., Houchen C.W, & Li M. (2022). Acetyl-coA Synthetase 2 potentiates macropinocytosis and muscle wasting through metabolic reprogramming in pancreatic cancer. Gastroenterology, 163(5), 1281-1293.e1.

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Western Blot Analysis of FoxO1 and MuRF1 Proteins

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Western blots were prepared essentially as described in [50 (link)]. Fifteen to thirty μg protein were reduced, denatured and electrophoretically separated on a 12% polyacrylamide gel with a 5.2% polyacrylamide stacking gel on top. Gels were electroblotted onto PVDF Plus transfer membranes (Amersham Hybond-P, GE Healthcare, Buckinghamshire, England) and the membranes were blocked and then incubated with antibodies. Primary antibodies for detecting total-FoxO1 (rabbit monoclonal) (C29H4) [2880] and pFoxO1 S256 (rabbit polyclonal) [9461] were obtained from Cell Signaling Technology (Beverly, CA), MuRF1 (goat polyclonal) [AF5366] was obtained from R&D systems (Abingdon, England) and Ac-FoxO1 (rabbit polyclonal) (FKHR D19) [49437] from Santa Cruz Biotechnology (Santa Cruz, CA). All primary antibodies were used at a dilution of 1/800 – 1/1500. Antibodies were visualized with horseradish peroxidise conjugated secondary immunoglobulin diluted 1/1000 goat anti-rabbit IgG [P0448] and 1/1000-1/10000 rabbit anti-goat IgG [P0449] (Dako, Glostrup, Denmark). The bound immune complexes were detected using the ECL Plus Western blotting detection system and Hyperfilm ECL (Amersham International and Amersham Pharmacia Biotech, Buckinghamshire, England).

Fjällström A.K., Evertsson K., Norrby M, & Tågerud S. (2014). Forkhead box O1 and muscle RING finger 1 protein expression in atrophic and hypertrophic denervated mouse skeletal muscle. Journal of Molecular Signaling, 9, 9.

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Assaying Protein Interactions and Modifications

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Cells were lysed in Cell Lysis Buffer (Cell Signaling), supplemented with protease (#11697498001; Roche) and phosphatase inhibitors (#04906845001; Roche), in addition to 0.2 M glycerol-2-phosphate (Sigma-Aldrich) and resolved on NuPAGE gels (Novex; Life Technologies). Primary antibodies used in this study are β-actin (1:10,000, Cat. #A5441; Sigma-Aldrich), CAP350 (1:1000, Cat. #20022-1-AP; Acris Antibodies, San Diego, CA, USA), FLAG-TRα (1:1000, anti-FLAG #F7425; Sigma-Aldrich), GAPDH (1:10,000, Cat. #G8795; Sigma-Aldrich), MuRF1 (1:250–1:1000, #AF5366; R&D Systems), GAPDH (1:4000, G8795; Sigma-Aldrich), p-Rb (#sc-50; Santa Cruz Biotechnology), myc-tagged MuRF1 (1:4000, anti-myc, #C4439; or anti-myc-HRP-linked primary, 1:10,000, #A5598; Sigma-Aldrich), glutathione-S-transferase (GST)-tagged TRα (anti-GST; #G1160; Sigma-Aldrich, 1:10,000), TRα (PA1-211A; Thermo Scientific, 1:500), and HA-ubiquitin (anti-HA; #sc-57592, 1:1000). HRP-linked secondary antibodies used in this study are anti-mouse (#NA931V; GE Healthcare Life Sciences, 1:10,000), anti-rabbit (#A9169, Sigma-Aldrich, 1:20,000), and anti-goat (#A5420; Sigma-Aldrich, 1:10,000).

Wadosky K.M., Berthiaume J.M., Tang W., Zungu M., Portman M.A., Gerdes A.M, & Willis M.S. (2016). MuRF1 mono-ubiquitinates TRα to inhibit T3-induced cardiac hypertrophy in vivo. Journal of molecular endocrinology, 56(3), 273-290.

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