The ChIP assays were performed according to previous studies with minor modifications (Hu et al., 2017 (link); Miao et al., 2006 (link)). Briefly, the primary hepatocytes transduced with Ad-shCp or Ad-shCon were cross-linked with 1% formaldehyde for 15 min, and 125 mM glycine was added for 5 min to quench the reaction. The cells were lysed with the lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH = 8.0) and sonicated eight times on ice (15 s ON, 90 s OFF). The supernatant was incubated overnight with the PGC1α (Abcam, UK: ab54481) or PPARα (Abcam, UK; ab24509) antibodies. After precipitation with protein A/G Sepharose beads, the fragmented DNA was pulled down and subjected to qPCR analysis. The sequences of the primers are listed in Table S3 .
Ab54481
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab54481 is a laboratory equipment product. It is a tool used for scientific research and experiments. The core function of this product is to facilitate specific tasks or measurements within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Ab10983, by Abcam (9 mentions)
Ab131607, by Abcam (9 mentions)
β actin, by Cell Signaling Technology (6 mentions)
Ab186735, by Abcam (5 mentions)
Ab24509, by Abcam (4 mentions)
Ab110413, by Abcam (4 mentions)
Ab110304, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (4 mentions)
Image lab software, by Bio-Rad (3 mentions)
Sirt1, by Cell Signaling Technology (3 mentions)
Ab9485, by Abcam (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Ab3508, by Abcam (3 mentions)
Sc 47778, by Santa Cruz Biotechnology (3 mentions)
Ab57602, by Abcam (3 mentions)
Ipvh00010, by Merck Group (3 mentions)
Sc17196, by Abcam (2 mentions)
Ab126599, by Abcam (2 mentions)
96 protocols using ab54481
1
ChIP Assay for Transcription Factors
2
Antibody-based Protein Expression Analysis
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3
Protein Expression Analysis Protocol
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Protein was extracted using a lysis buffer (Sigma cat#C3228) containing a cocktail of phosphatase (Sigma cat#4906845001) and protease inhibitors (Sigma cat#P2714–1BTL). After homogenizing the tissues, the samples were centrifuged at 16.1 relative centrifugal force (rcf) for 15 min at 4 °C. The middle layer was transferred to a prechilled tube. Protein levels of mTOR, phosphorylated protein kinase B (PKB, also known as AKT), phosphorylated insulin receptor substrate-1 (IRS-1), PGC-1α, and glycerol kinase (GyK) were measured by immunoblotting. Total 15 μg of protein was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and all of the separated proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk for 1 h at room temperature (RT) and exposed to the primary anti-bodies overnight at 4 °C (mTOR, Cell signaling cat#2983; pAKT, Cell signaling cat#9271; pIRS-1, Santa Cruz cat#sc17196; PGC-1α, Abcam cat#ab54481; and GyK, Abcam cat#ab126599). Then, the secondary anti-bodies were exposed for 1 h at RT. Finally, chemiluminescent substrate was applied, and bands of the protein were visualized by Molecular Imager Gel DocTM XR system. Each protein expression was normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, or total AKT and quantified using ImageJ software from the NIH.
4
Western Blot Analysis of Protein Targets
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Western Blotting was carried out using specific antibodies; Bcl-2 (D17C4, Cell Signaling, France), Bax (ab115193, Abcam, France), aconitase 2 (ab110321, Abcam, France), nitrotyrosine (05-233, Millipore, France), 4HNE (ab5605, Millipore, France), sirtuine-3 (D22A3, Cell signaling, France), sirtuine-1 (SC-9475, Cell signaling, France), phosphorylated sirtuine-1 (Sc-2314, Cell signaling, France), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a, ab54481, Abcam, France), nuclear factor (erythroid-derived 2)-like 2 (NRF2, ab31163, Abcam, France), mammalian target of rapamycin (mTOR, 2983S, Cell signaling, France), phospho mTOR (5536P, Cell signaling, France), eNOS total (610296, BD Transduction, Le Pont de Claix, France) and eNOS phophorylated Ser1177 (C9C3; Cell-Signaling, Saint Quentin-Yvelines, France). Protein quantification level was expressed as ratio of total proteins loaded on precast gels with stain-free technology (BioRad, France). All blots were obtained by imaging with chemidoc and quantificated with ImageLab software (BioRad).
5
Immunohistochemistry for Nrf2 and PGC-1α
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The paraffin sections (5 mm thickness) were deparaffinaged in xylene, and then rehydrated with various grades of ethanol (100, 95, 90, 80 and 70%). The sections were exposed to 3% H2O2 for 10 min at 37°C, and treated with citrate-buffered saline for 15 min at 95°C. By incubating the sections in 10% bovine serum albumin, non-specific binding of immunoglobulins was blocked for 10 min. Subsequently, the sections were incubated overnight at 4°C with primary antibodies, Nrf2 (ab31163, 1:1,000 dilution) or PGC-1α (ab54481, 1:1,000 dilution) (both from Abcam, Cambridge, MA, USA) and then rinsed with PBS and incubated for 1 h with peroxidase-conjugated secondary antibody. The sections were visualized with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining. Finally, the stained sections were examined under a fluorescence microscope (Olympus).
6
Molecular Mechanisms of Adipocyte Metabolism
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Rb1 (>98%, ab142646) was purchased from Abcam (Cambridge, UK). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex), insulin, and Oil Red O powder were purchased from Sigma (St. Louis, MO, United States). L748337 was from Tocris Bioscience (Bristol, UK). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Corning (NY, United States). Antibodies for liver kinase B1 (LKB1) (3047S), pLKB1 (Ser428) (3482S), AMP-activated protein kinase alpha (AMPKα) (2532S), pAMPKα (Thr172) (2535S), acetyl-CoA carboxylase (ACC) (3676S), pACC (Ser79) (3661S), silent information regulator T1 (SIRT1) (8469S), SIRT3 (5490S), phospho-hormone sensitive lipase (pHSL) (Ser563) (4139S), phospho-PKA substrate (9624S), UCP1 (14670S), and β-actin (3700S) were purchased from Cell Signaling Technology (Beverly, MA, United States); the antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-32233) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States); antibodies for PGC1α (ab54481), comparative gene identification 58 (CGI58) (ab59488), adipose triglyceride lipase (ATGL) (ab207799), HSL (ab45422), and β3AR (ab94506) were purchased from Abcam (Cambridge, UK); the antibody for PPARα (PA1-822A) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
7
Histological Analysis of Tissue Sections
8
Whole Mount Immunofluorescence Experiments
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Whole mount IF experiments were completed as previously described (Gerlach and Wingert, 2014 (link); Kroeger et al., 2017 (link); Marra et al., 2017 , 2019c (link)). For cilia and basal bodies, anti-tubulin acetylated diluted 1:400 (Sigma T6793) and anti γ-tubulin diluted 1:400 (Sigma T5192) were used, respectively. The anti-Ppargc1a was diluted 1:150 (Abcam, AB54481) (Ran et al., 2017 (link), Blechman et al., 2011 (link)), and anti-PKC was diluted 1:500 (Santa Cruz, SC216). Anti-rabbit and anti-mouse secondary antibodies were diluted 1:500 (Alexa Fluor, Invitrogen).
9
Transcriptional Analysis of Metabolic Enzymes
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Total RNA from cultured cells was extracted using Trizol reagent (Invitrogen). cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Real-time qPCR was performed using the following TaqMan gene expression assays (Thermo Fisher Scientific): L2HGDH (Hs00227575), PPARGC1A (Hs00173304_m1), MDH1 (Hs00936497_g1), MDH2 (Hs00938918_m1), ACO2 (Hs00426616_g1) and OGDH (Hs01081865_m1). HPRT1 (Hs02800695_m1) and RPLPO (hs99999902_m1) probes were used as an internal control, and the ΔΔCt method was used to calculate relative mRNA levels. For immunoblotting, anti-α-L2HGDH (GeneTex, GTX32695, 1:2000; and Novus, NBP2-85197, 1:1000), anti-PGC-1α (Abcam, ab54481, 1:1000) anti-α-MDH2 (Abcam, ab96193, 1:3000), anti-MDH1 (Novus, NBP1-895151, 1:3000) and anti-β-actin (Abcam, ab20272, 1:3000) were used as per the manufacturers' instructions.
10
Quantification of PGC-1α and SIRT3 in Neurons
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The samples from primary neurons in vitro and cerebral cortices in vivo were lysed with RIPA buffer (Beyotime, Jiangsu, China). After protein concentrations were measured, the same amount of protein from every sample was separated and transferred. After blocking with defatted milk, the membranes were hatched with primary antibodies overnight at 4°C against PGC-1α (1: 2000, ab54481; Abcam, Cambridge, MA, USA), SIRT3 (1: 1000, ab86671; Abcam), and β-actin (1: 5000, AP0060; Bioworld Technology, Minneapolis, MN, USA). After washing with PBS containing Tween-20 (PBST), the membranes were incubated with appropriate secondary antibodies at room temperature. After incubation of the chemiluminescence solution, the protein bands were visualized. Band density was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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