The isolated liver samples were homogenized in ice-cold RIPA buffer containing protease and phosphatase inhibitors (Beyotime Biotechnology, China) to extract total protein, which concentration was determined with the BCA Protein Assay Kit (Beyotime Biotechnology). The aliquots of protein samples (20μg) were separated by 10% SDS-PAGE gel and then transferred to activated polyvinylidene fluoride membranes (Merck Millipore, Germany). After being blocked with 5% skimmed milk in TBST buffer (Sangon Biotech, China), the membranes were incubated with the primary antibody overnight in a refrigerator at 4°C and then with secondary antibody for 2h. The immune complexes were visualized with a Beyo ECL Plus kit (Beyotime Biotechnology) and quantified using ImageJ software (Bethesda, United States). The specific primary antibodies are anti-FoxO1 (ET1608-25, HuaBio, China), anti-phospho-FoxO1 (#9461, Cell Signaling Technology, United States) anti-Akt1 (#4691, Cell Signaling Technology, United States), anti-phospho-Akt1 (#4060, Cell Signaling Technology, United States), anti-Sirt1 (sc-74,465, Santa Cruz Biotechnology), anti-Nrf2 (ab62352, Abcam, China) and anti-β-tubuliin (#2146, Cell Signaling Technology, United States) antibodies. The secondary antibodies are anti-mouse antibody (Solarbio, China) and anti-rabbit antibody (SongonBiotech, China).
Anti sirt1 sc 74 465
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Sirt1 (sc-74,465) is a lab equipment product offered by Santa Cruz Biotechnology. It is an antibody that targets the Sirt1 protein. The core function of this product is to detect and analyze the Sirt1 protein in research samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt1, by Cell Signaling Technology (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Anti phospho akt1, by Cell Signaling Technology (1 mentions)
Anti phospho foxo1, by Cell Signaling Technology (1 mentions)
Anti mouse antibody, by Solarbio (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Tbst buffer, by Sangon (1 mentions)
Ez chip kit, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse, by Santa Cruz Biotechnology (1 mentions)
Luminatatm crescendo, by Merck Group (1 mentions)
3 protocols using anti sirt1 sc 74 465
1
Protein Extraction and Western Blotting
2
Chromatin Immunoprecipitation (ChIP) Assay
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The ChIP assay was performed using the EZ-ChIP kit (Millipore Corp., Billerica, MA, USA). In brief, the cells were treated with 37% methanol for protein crosslinking and then treated with 0.125 M glycine to terminate the reaction. After that, the chromatin-protein complexes were reacted with 5 μg anti-TCF3 (sc-416, 1:500, Santa Cruz Biotechnology) and anti-SIRT1 (sc-74465, 1:300, Santa Cruz Biotechnology) for immunoprecipitation. Thereafter, the abundance of promoter fragments in the immunoprecipitated complexes was examined using qPCR [29 (link)].
3
Western Blot Analysis of SIRT-1 and β-Actin
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In western blotting procedure equal amounts of cell extract were subjected to electrophoresis in 10% gels, blotted onto nitrocellulose membranes essentially as previously described 25, 26 , and probed with primary antibody at 4 C overnight. The following antibodies were used: anti-b-actin (A5316, SigmaeAldrich), anti-SIRT-1 (sc-74465, Santa Cruz Biotechnology). After washes, membranes were incubated with horseradish peroxidase-conjugated anti-mouse (Santa Cruz Biotechnology) IgG for 1 h. The chemiluminescent signals were detected using an ECL system (LuminataTM Crescendo, Millipore). b-actin was used as loading control. A representative image of visualized immunoreactive bands and densitometric analysis show the relative intensity of protein expression.
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