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Chrebp

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ChREBP is a transcription factor that regulates the expression of genes involved in glucose and lipid metabolism. It plays a key role in the regulation of carbohydrate and lipid homeostasis.

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Lab products found in correlation

Hnf4a, by Santa Cruz Biotechnology (2 mentions) Acetylated histone 4 ac h4, by Merck Group (2 mentions) Pcr clean up extraction kit, by Macherey-Nagel (2 mentions) Acetylated histone 3 ac h3, by Merck Group (2 mentions) Normal rabbit immunoglobulin g igg antibody, by Santa Cruz Biotechnology (2 mentions) Glut2, by Santa Cruz Biotechnology (2 mentions) Glut5, by Santa Cruz Biotechnology (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) P ampk, by Cell Signaling Technology (1 mentions) Nuclear extraction kit, by Abcam (1 mentions) Methyl pp2a c, by Santa Cruz Biotechnology (1 mentions) Pp2a b, by Cell Signaling Technology (1 mentions) Pp2a c, by Cell Signaling Technology (1 mentions) Image lab upgrade for chemidoc xrs system 170 8299, by Bio-Rad (1 mentions) Pf 4708671, by Selleck Chemicals (1 mentions) Lamin b1, by Thermo Fisher Scientific (1 mentions) α tubulin, by Merck Group (1 mentions) P src, by Cell Signaling Technology (1 mentions) 2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions) P akt, by Cell Signaling Technology (1 mentions)

12 protocols using chrebp

1

Genome-wide ChIP profiling of mouse liver

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In vivo chromatin immunoprecipitation (ChIP) assays from mouse livers were performed by Active Motif. Briefly, genomic DNA regions of interest were isolated using antibodies against H3K9Ac (Active Motif), RNA Pol II (Active Motif), ChREBP (Novus), and PPARα (Santa Cruz Biotechnology). qPCR reactions were carried out in triplicate using SYBR Green Supermix (Bio-Rad) on a CFX Connect Real Time PCR system. Positive and negative control sites were tested for each factor as well as the sites of interest. The resulting signals were normalized for primer efficiency by carrying out qPCR for each primer pair using input DNA (pooled unprecipitated genomic DNA from each sample). Specific enrichment was expressed as percentage of input. Further details are provided in Supplemental Experimental Procedures.
In vitro ChIP assays from cultured hepatocytes were performed as described previously (Marmier et al., 2015 (link)). Briefly, genomic DNA regions of interest were isolated using antibodies against ChREBP (Novus) and immunoglobulin G (IgG) (Cell Signaling). DNA fragments were quantified by qPCR using primers described in Table S1. Results are expressed as fold enrichment. Further details are provided in Supplemental Experimental Procedures.
Iroz A., Montagner A., Benhamed F., Levavasseur F., Polizzi A., Anthony E., Régnier M., Fouché E., Lukowicz C., Cauzac M., Tournier E., Do-Cruzeiro M., Daujat-Chavanieu M., Gerbal-Chalouin S., Fauveau V., Marmier S., Burnol A.F., Guilmeau S., Lippi Y., Girard J., Wahli W., Dentin R., Guillou H, & Postic C. (2017). A Specific ChREBP and PPARα Cross-Talk Is Required for the Glucose-Mediated FGF21 Response. Cell Reports, 21(2), 403-416.
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2

Quantifying Hepatic Nuclear Proteins

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Nuclear proteins were extracted from livers with Nuclear Extraction Kit (Abcam, Cambridge, MA). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc., Waltham, MA). Western blot was performed as described previously [29] (link) to detect PPARα (peroxisome proliferator-activated receptor-α), CPT1 (Carnitine palmitoyltransferase I), SREBP (Sterol-regulatory element binding protein), ADPN receptor 1, ADPN receptor 2, methyl-PP2A-C(Santa Cruz Biotechnologies, Santa Cruz, CA), FAS (fatty acid synthetase), SCD1 (Stearoyl-CoA desaturase-1), PP2A (protein phosphatase 2A) A, PP2A B, PP2A C, pAMPK, LKB1 (Cell Signaling Technologies, Beverly, MA), and ChREBP (Novus Biologicals, Littleton, CO). Blots were scanned using a Bio-Rad Imaging System (Image Lab™ Upgrade for ChemiDoc™ XRS + System #170-8299). All specific bands were quantified with the Automated Digitizing System (Image Lab 4.1). Results are representative of three independent experiments.
Zhao C., Liu L., Liu Q., Li F., Zhang L., Zhu F., Shao T., Barve S., Chen Y., Li X., McClain C.J, & Feng W. (2019). Fibroblast growth factor 21 is required for the therapeutic effects of Lactobacillus rhamnosus GG against fructose-induced fatty liver in mice. Molecular Metabolism, 29, 145-157.
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3

ChIP Analysis of Liver Histone Modifications

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Chromatin immunoprecipitation (ChIP) analysis was performed as described,23 with the following modifications. Before cross‐linking with 1% formaldehyde, livers were homogenized in PBS and cross‐linked with 0.5 M of di(N‐succinimidyl) glutarate for 45 minutes at room temperature. Immunoprecipitation of samples was performed overnight at 4°C with 3 µg of ChREBP (Novus), acetylated histone 4 (Ac‐H4; Millipore), acetylated histone 3 (Ac‐H3; Millipore), HNF4A (Santa Cruz), or normal rabbit immunoglobulin G (IgG) antibody (Santa Cruz). DNA was purified using the PCR Clean‐up Extraction Kit (Macherey‐Nagel), after which qPCR was performed. Primers are listed in Supporting Table S7.
Hoogerland J.A., Lei Y., Wolters J.C., de Boer J.F., Bos T., Bleeker A., Mulder N.L., van Dijk T.H., Kuivenhoven J.A., Rajas F., Mithieux G., Haeusler R.A., Verkade H.J., Bloks V.W., Kuipers F, & Oosterveer M.H. (2019). Glucose‐6‐Phosphate Regulates Hepatic Bile Acid Synthesis in Mice. Hepatology (Baltimore, Md.), 70(6), 2171-2184.
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4

Molecular Mechanisms of Cellular Stress

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FMK and BI-D1870 were purchased from Axon Medchem (Groningen, The Netherlands). PP2 was purchased from Cayman Chemical (Ann Arbor, MI, USA). PF-4708671 was purchased from Selleck Chemicals (Houston, TX, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). TM, D-(+)-glucose and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from the following vendors: TXNIP (MBL International, Woburn, MA, USA); ChREBP (Novus Biologicals, Centennial, CO, USA); KDEL (GRP94, GRP78) (Enzo Life Sciences, Lörrach, Germany); CREB-2 (ATF4), GADD153 (CHOP) and Src (Santa Cruz, Santa Cruz, CA, USA); Akt, p-Akt, PARP, cleaved Caspase-3, p-Src, p-S6, S6, ACC, and FAS (Cell Signaling Technology; Danvers, MA, USA); Lamin B1 (Invitrogen); α-tubulin (Sigma-Aldrich).
Han J.H., Kim S., Kim S., Lee H., Park S.Y, & Woo C.H. (2019). FMK, an Inhibitor of p90RSK, Inhibits High Glucose-Induced TXNIP Expression via Regulation of ChREBP in Pancreatic β Cells. International Journal of Molecular Sciences, 20(18), 4424.
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5

Chromatin Immunoprecipitation Assay with Modifications

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Chromatin immunoprecipitation (ChIP) analysis was performed as described,(23 (link)) with the following modifications. Before cross-linking with 1% formaldehyde, livers were homogenized in PBS and cross-linked with 0.5 M of di(N-succinimidyl) glutarate for 45 minutes at room temperature. Immunoprecipitation of samples was performed overnight at 4°C with 3 μg of ChREBP (Novus), acetylated histone 4 (Ac-H4; Millipore), acetylated histone 3 (Ac-H3; Millipore), HNF4A (Santa Cruz), or normal rabbit immunoglobulin G (IgG) antibody (Santa Cruz). DNA was purified using the PCR Clean-up Extraction Kit (Macherey-Nagel), after which qPCR was performed. Primers are listed in Supporting Table S7.
Hoogerland J.A., Lei Y., Wolters J.C., de Boer J.F., Bos T., Bleeker A., Mulder N.L., van Dijk T.H., Kuivenhoven J.A., Rajas F., Mithieux G., Haeusler R.A., Verkade H.J., Bloks V.W., Kuipers F, & Oosterveer M.H. (2019). Glucose‐6‐Phosphate Regulates Hepatic Bile Acid Synthesis in Mice. Hepatology (Baltimore, Md.), 70(6), 2171-2184.
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6

Immunohistochemical Analysis of Intestinal Proteins

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Small intestine specimens were fixed in neutral-buffered formalin and embedded in paraffin blocks according to standard procedures. The paraffin-embedded tissue sections were incubated in the oven at 60°C for 1 h. After rehydration in graded ethanol, antigen retrieval was performed in 1× Tris-EDTA, pH 9.5 (Thermo Scientific, Boston, MA) by heating in a microwave for 10 min. Sections were incubated with 3% H2O2 (Dako, Denmark) at room temperature for 30 min, blocked for 5 min, and then incubated with the primary antibody overnight. The next day, sections were incubated with the secondary antibody for 30 min, washed with DPBS containing Tween 20, and stained with 3,3′-diaminobenzidine. Primary antibodies against the following proteins were used: ChREBP (Novus Biologicals, Littleton, CO), GLUT5 (Santa Cruz Biotechnology, Santa Cruz CA), GLUT2 (Santa Cruz Biotechnology, Santa Cruz CA), and NHE3 (Santa Cruz Biotechnology, Santa Cruz CA).
Oh A.R., Sohn S., Lee J., Park J.M., Nam K.T., Hahm K.B., Kim Y.B., Lee H.J, & Cha J.Y. (2018). ChREBP deficiency leads to diarrhea-predominant irritable bowel syndrome. Metabolism: clinical and experimental, 85, 286-297.
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7

Antibody Profiling for Multimodal Analysis

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The following antibodies were used in this study for either western blotting, ChIP-sequencing, immunohistochemistry and chromatin immunoprecipitation. SOD2 (The Binding site, PC096), Gpx1 and Gpx4 (gift from L. Chavatte), ChREBP (Novus, NB400), SCD1 (Cell Signaling, C12H5), Phf2 (Cell Signaling, D45A2), H3K9me1 (Abcam, ab8896), H3K9me2 (Abcam, ab1220), H3K9me3 (Abcam, ab8898), histone H3 (Diagenode, C15200011), p(S473)-AKT (Cell signaling, D9E), Akt (Cell Signaling, 9272), P-p70S6K (Cell Signaling, 108D2), p70S6K (Cell Signaling, 49D7), p-GSK3β (Cell Signaling, D17D2), GSK3β (Cell Signaling, 27C10), LPK (Abcam, ab6191), ACC (Cell Signaling, 3662), FAS (Cell signaling, C20G5), p62 (gift from A. F. Burnol), Nrf2 (Santa Cruz Biotechnology, 13032), G6PDH (Cell Signaling, 12263), TKT (Cell Signaling, 8616), α-SMA (Novus, NB-600-531), collagen I (Novus, NB600-408), GAPDH (Santa Cruz Biotechnology, FL-335), malic enzyme (Novus, 68578), MTTP (Novus, 62489), NQO1 (Novus, A180), RNA PolII (Santa Cruz, sc899), HSP90 (Cell Signaling, 4874), Cidec (Abcam, ab77115), Plin2 (Progen, ap125), G0S2 (Santa Cruz Biotechnology, N13), and FLAG (Sigma, F1804).
Bricambert J., Alves-Guerra M.C., Esteves P., Prip-Buus C., Bertrand-Michel J., Guillou H., Chang C.J., Vander Wal M.N., Canonne-Hergaux F., Mathurin P., Raverdy V., Pattou F., Girard J., Postic C, & Dentin R. (2018). The histone demethylase Phf2 acts as a molecular checkpoint to prevent NAFLD progression during obesity. Nature Communications, 9, 2092.
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8

Western Blot Analysis of Adipocyte Markers

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Cells were lysed in radioimmunoprecipitation assay buffer [50 mM tris(hydroxymethyl)aminomethane, pH 8.0/150 mM NaCl/1.0% octylphenoxypolyethoxyethanol/0.5% deoxycholate/0.1% sodium dodecyl sulfate/0.2 mM NaVO4/10 mM NaF/0.4 mM EDTA/10% glycerol with protease inhibitors] (Roche). Lysates were sonicated for 20 seconds on ice and centrifuged at 10,000g for 5 minutes. Lysates were then placed in a boiling water bath for 5 minutes with Laemmli loading buffer, followed by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels. Western blot analyses were performed with antibodies directed at Ucp1 (Sigma Aldrich), ChREBP (Novus Biologicals, Littleton, CO), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, MA) at the recommended antibody dilutions. The blots were scanned with the LI-COR laser-based image detection method.
Katz L.S., Xu S., Ge K., Scott D.K, & Gershengorn M.C. (2017). T3 and Glucose Coordinately Stimulate ChREBP-Mediated Ucp1 Expression in Brown Adipocytes From Male Mice. Endocrinology, 159(1), 557-569.
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9

Western Blotting of ER Stress Markers

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Western blotting was performed using primary antibodies against phosphorylated PERK, phosphorylated EIF2α, EIF2α, CHOP, C/EBPβ, GAPDH, β-actin (Cell Signaling, MA, USA), ATF6 (Abcam, Maharashtra, India), FAS (BD Bioscience, NJ, USA), SREBP-1c (Thermo Scientific, NH, USA), carbohydrate-responsive element-binding protein (CHREBP) (Novus Biologicals, NJ, USA), and LaminB (Santa Cruz Biotechnology, CA, USA). Western blots were developed using ECL Western blotting substrate and quantified using ImageJ software (for results, see Supplementary Fig. 10).
Zhang Z., Li B., Meng X., Yao S., Jin L., Yang J., Wang J., Zhang H., Zhang Z., Cai D., Zhang Y, & Ning G. (2016). Berberine prevents progression from hepatic steatosis to steatohepatitis and fibrosis by reducing endoplasmic reticulum stress. Scientific Reports, 6, 20848.
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10

Protein Expression Analysis in Hepatocytes

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Proteins from hepatocytes and liver tissue were extracted from whole-cell lysates. Proteins were subjected to 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Rabbit polyclonal ChREBP (1:1,000, Novus Biologicals) and L-PK (1:1,500, a gift from Dr. Axel Kahn) antibodies were used. Protein β-actin (1:5,000) (Cell Signaling Technology) was used to normalize data.
Iroz A., Montagner A., Benhamed F., Levavasseur F., Polizzi A., Anthony E., Régnier M., Fouché E., Lukowicz C., Cauzac M., Tournier E., Do-Cruzeiro M., Daujat-Chavanieu M., Gerbal-Chalouin S., Fauveau V., Marmier S., Burnol A.F., Guilmeau S., Lippi Y., Girard J., Wahli W., Dentin R., Guillou H, & Postic C. (2017). A Specific ChREBP and PPARα Cross-Talk Is Required for the Glucose-Mediated FGF21 Response. Cell Reports, 21(2), 403-416.
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