Sirt1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China, Japan

SIRT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase enzyme. It plays a key role in the regulation of cellular processes by deacetylating target proteins.

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Lab products found in correlation

264 protocols using sirt1

Western Blot Analysis of Cellular Proteins

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Twenty micrograms of cell lysate were analyzed on a denaturing 4%-15%
gradient polyacrylamide gel (Biorad, 567–1081, www.bio-rad.com), transferred onto a PVDF membrane (Millipore,
IPVH08100, www.merckmillipore.com) and incubated with various antibodies
purchased from Cell Signaling Technology, which include anti-Vav1 (Cat. 4657),
Sirt1 (Cat. 3931), Sirt1 (Cat. 8469S, for human), PPARγ (Cat. 2443),
C/EBPα (Cat. 2295), Creb (Cat. 9197S), phospho-Creb (Cat. 9191S), and
acetylated lysine (Cat. 9441). Antibody against Col2α1 was purchased from
Santa Cruz Biotechnology (Cat. sc-52658). All antibodies were used at 1:500
dilution unless otherwise specified. Immune membranes were visualized using
horseradish peroxidase-conjugated secondary antibodies (Cell signaling, Cat 7074
and 7076, www.cellsignal.com) and a chemiluminescent
substrate (ECL system, GE Healthcare, www.gelifesciences.com).

Qu P., Wang L., Min Y., McKennett L., Keller J.R, & Lin P.C. (2016). Vav1 Regulates Mesenchymal Stem Cell Differentiation Decision Between Adipocyte and Chondrocyte via Sirt1. Stem cells (Dayton, Ohio), 34(7), 1934-1946.

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Quantification of AMPK, p53, and SIRT1 Signaling

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Total AMPK, phosphorylation of AMPK (pAMPK), AcP53, FOXO1, and SIRT1 were assayed by Western blot analysis. Twenty micrograms (for AMPK, pAMPK, and FOXO1), 10 μg (for AcP53), and 40 μg (for SIRT1) of protein were loaded, and antibodies (AMPK, pAMPK, FOXO1, SIRT1 (Cell Signaling, Danvers, Mass. USA) and AcP53 (Abcam, Cambridge, Mass. USA)) were used in 1:1000 (AMPK, pAMPK, and SIRT1) and 1:2000 (AcP53, FOXO1) concentrations.

Toklu H.Z., Scarpace P.J., Sakarya Y., Kirichenko N., Matheny M., Bruce E.B., Carter C.S., Morgan D, & Tümer N. (2016). Intracerebroventricular tempol administration in older rats reduces oxidative stress in the hypothalamus but does not change STAT3 signalling or SIRT1/AMPK pathway. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme, 42(1), 59-67.

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Immunoprecipitation and Acetylation Analysis

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Brain tissue from lesioned cortices were incubated with 1 μg of SIRT1 (Cell Signaling Technology) or HMGB1 anti-acetylated lysine antibody (Cell Signaling Technology) for 2 h at 4 °C. A 10-μl volume of protein A/G agarose beads (Roche, Mannheim, Germany) was added to the samples and incubated overnight. After immunoprecipitation and centrifugation, agarose beads were washed three times with lysis buffer, and the degree of acetylation of SIRT1 or HMGB1 was analyzed by western blotting using an anti-acetylated lysine antibody (Cell Signaling Technology).

Chen X., Chen C., Fan S., Wu S., Yang F., Fang Z., Fu H, & Li Y. (2018). Omega-3 polyunsaturated fatty acid attenuates the inflammatory response by modulating microglia polarization through SIRT1-mediated deacetylation of the HMGB1/NF-κB pathway following experimental traumatic brain injury. Journal of Neuroinflammation, 15, 116.

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Western Blot Analysis of Cellular Proteins

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Cells or tissues were lysed in RIPA Buffer (Solarbio, Beijing, China) containing proteinase inhibitor cocktail. Proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated with primary antibodies including CitH3 (1:1000), MPO (1:1000), LC3B (1:1000), SQSTM1/p62 (1:1000), GPX4 (1:2000, ab125066, Abcam), cleaved caspase-3 (1:1000, 9661, Cell Signaling Technology), caspase-3 (1:1000, 9662, Cell Signaling Technology), caspase-11 (1:1000, 14340, Cell Signaling Technology), SIRT1 (1:1000), METTL3 (1:1000), β-Actin (1:3000, 3700, Cell Signaling Technology). Signals were detected with a ECL chemiluminescence kit (Tanon, Shanghai, China) after HRP-conjugated secondary antibodies incubation.

Qu M., Chen Z., Qiu Z., Nan K., Wang Y., Shi Y., Shao Y., Zhong Z., Zhu S., Guo K., Chen W., Lu X., Wang Z., Zhang H, & Miao C. (2022). Neutrophil extracellular traps-triggered impaired autophagic flux via METTL3 underlies sepsis-associated acute lung injury. Cell Death Discovery, 8, 375.

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Western Blot Protocol for Protein Analysis

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The Western blot analysis was performed as reported previously^{14 (link)}. Total cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 1 h, and incubated with the primary antibodies listed next for 1 h with shaking. The primary antibodies against TG2, β-catenin, p-β-catenin, non-p-β-catenin, Ampka, p-Ampka, Foxo3a, p-Foxo3a, p65, p-p65, mTOR, p-mTOR, Axin2, Tcf1, Sirt1, and IκB were purchased from Cell Signaling Technology (Danvers, MA); antibodies against Sox9, Runx2, Mmp3, Mmp13, and Adamts5 were from Abcam (Cambridge, UK); the antibodies against LaminB and haemagglutinin (HA) were from Santa Cruz Biotechnology (Dallas, TX), and β-actin antibody was from Sigma Aldrich (St. Louis, MO). Membranes were washed, and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. The membrane signal was visualized using Supersignal Chemiluminescent Substrates (Thermo Fisher Scientific, Waltham, MA).

Han M.S., Jung Y.K., Kim G.W, & Han S. (2020). Transglutaminase-2 regulates Wnt and FoxO3a signaling to determine the severity of osteoarthritis. Scientific Reports, 10, 13228.

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Carfilzomib Modulates Apoptosis Pathways

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Carfilzomib (CFZ) was purchased from Onyx Pharmaceuticals (San Francisco, CA, USA). Resveratrol (RSV), N-Acetylcysteine (NAC), methyl-thiazolyl tetrazolium (MTT), 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe, and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA). 3-methyladenine (3-MA) was obtained from Abmole inhibitor leader (Houston, TX, USA). The CFZ and NAC were dissolved in double-distilled water, and the RSV and 3-MA were dissolved in DMSO, respectively. Primary antibodies of SIRT1 (Cat. 2310), Smac (Cat.15107), survivin (Cat.2808), p-p38 (Cat.4511), p53 (Cat.2527), PARP (Cat.9542), caspase 3 (Cat.9662), LC3-I/II (Cat. 3868), and tubulin (Cat. 2144) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against tubulin was from Wuhan Boster Biological Technology (Wuhan, Hubei, China). Scrambled siRNA AND SMARTpool: ON-TARGETplus DIABLO (Smac) siRNA were purchased from Dharmacon.

Li Q., Yue Y., Chen L., Xu C., Wang Y., Du L., Xue X., Liu Q., Wang Y, & Fan F. (2018). Resveratrol Sensitizes Carfilzomib-Induced Apoptosis via Promoting Oxidative Stress in Multiple Myeloma Cells. Frontiers in Pharmacology, 9, 334.

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Molecular Mechanisms of Muscle Atrophy

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UA and Ex-527 were acquired from MedChemExpress (Princeton, NJ, USA). The primary antibody against myosin heavy chain (MyHC, MAB4470) was acquired from R&D Systems (Minneapolis, MN, USA). The primary antibodies against STAT3 (#9139), p-STAT3 (Yyr705) (#9145), NF-κB (p65) (#8242), p-NF-κB (p-p65) (Ser536) (#3033), and SIRT1 (#9475) were acquired from Cell Signaling Technology (Beverly, MA, USA); FOXO3a (#10849-1-Ap), FOXO1 (#18592-1-Ap), NOX4 (#14347-1-Ap), and GAPDH (#10494-1-Ap) were acquired from Proteintech (Wuhan, China); and Atrogin-1 (ab168372) and MuRF1 (ab172479) were acquired from Abcam (Cambridge, MA, USA).

Tao W., Ouyang Z., Liao Z., Li L., Zhang Y., Gao J., Ma L, & Yu S. (2023). Ursolic Acid Alleviates Cancer Cachexia and Prevents Muscle Wasting via Activating SIRT1. Cancers, 15(8), 2378.

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Western Blot Analysis of Protein Expression

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hVSMCs and tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime) and centrifuged at 4°C to extract total protein. Protein concentration was detected using a bicinchoninic acid (BCA) kit (Bio-Rad). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with Bcl-2 (cat. no. 4223; 1: 1000; Cell Signaling Technology, Inc.), Bax (cat. no. 5023; 1: 1000; Cell Signaling Technology, Inc.), MMP9 (cat. no. 13,667; 1: 1000; Cell Signaling Technology, Inc.), SIRT1 (cat. no. 2496; 1: 1000; Cell Signaling Technology, Inc.), or GAPDH (cat. no. 5174; 1: 1000; Cell Signaling Technology, Inc.) antibody at 4°C overnight after blocking with 5% nonfat milk phosphate buffer saline (PBS)-0.1%Tween 20 solution for 1 h. The membrane was then incubated with secondary antibodies (cat. no. ab96899; 1:2,000; Abcam, Cambridge, USA) for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence luminescent substrate (Amersham ImageQuant800UV; Cytiva, MA, USA) and quantified using ImageJ software.

Yuan L., Wang D, & Wu C. (2022). Protective effect of liquiritin on coronary heart disease through regulating the proliferation of human vascular smooth muscle cells via upregulation of sirtuin1. Bioengineered, 13(2), 2840-2850.

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Western Blot Analysis of Autophagy Markers

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Colon samples were homogenized on ice in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing phenylmethylsulfonyl fluoride and protease inhibitor. The protein concentrations of the samples were determined using the Bradford method (using a Bio-Rad Protein assay; Bio-Rad Laboratories, Inc.). Equivalent amounts of protein (50 µg) from each sample were separated on 12% sodium dodecyl sulfate-polyacrylamide gels, and fractioned proteins were transferred onto 0.22 µM nitrocellulose membranes (EMD Millipore, Billerica, MA, USA) at 75 V for 45 min. Subsequently, the membranes were blocked with Tris-buffered saline solution containing 5% nonfat dried milk at room temperature for 1 h, and then incubated with specific antibodies against Atg12 (cat. no. 2011), Beclin-1 (cat. no. 3495), LC3II (cat. no. 4108), mTOR (cat. no. 2972) and SIRT1 (cat. no. 8469; all 1:1,000 dilution; all from Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. Bands were visualized using a chemiluminescence detection system (cat. no. #34095; Pierce; Thermo Fisher Scientific, Inc.) and quantified with Quantity One Software 4.6.7 (Bio-Rad Laboratories, Inc.).

Zhang L., Xue H., Zhao G., Qiao C., Sun X., Pang C, & Zhang D. (2019). Curcumin and resveratrol suppress dextran sulfate sodium-induced colitis in mice. Molecular Medicine Reports, 19(4), 3053-3060.

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Immunofluorescence Assay for Parkin and Sirt1

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A total of 1 × 10⁶ cells were seeded into the culture dish, washed with PBS, and permeabilized with PBS containing 0.1% Triton X-100 and 0.1% sodium citrate at 4°C. Next, the samples were blocked with 10% goat serum albumin for 1 h and cultured at 4°C overnight with the primary antibodies Parkin (1/1,000, Cell Signaling Technology, Beverly, MA, USA) and Sirt1 (1/1,000, Cell Signaling Technology). Following PBS washing three times, the samples were incubated with Alexa Fluor 488 donkey anti-rabbit antibody (1/1,000, Invitrogen) for 1 h. Cells were observed under an inverted fluorescence microscope at 40× magnification (BX51, Olympus).

Guo J., Wang R, & Liu D. (2021). Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate Sepsis-Induced Acute Kidney Injury by Promoting Mitophagy of Renal Tubular Epithelial Cells via the SIRT1/Parkin Axis. Frontiers in Endocrinology, 12, 639165.

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