Whole-tissue or whole-cell proteins were isolated and homogenized by standard methods and separated by sodium dodecyl suphate–polyacrylamide gel electrophoresis. Protein concentrations were determined by the BCA method (Thermo Scientific). After incubation with antibodies for RetSat5 (link) (diluted 1:1000 in 4% skimmed milk), ChREBP (NB400-135, lot J1,M1 or M6 (Novus Biologicals, diluted 1:1000 in 0.05% BSA)25 (link) (both antibodies were validated in the referenced studies), Ran (#610340, BD Biosciences, diluted 1:1000 in 4% skimmed milk), Anti-flag (A8592, Sigma, diluted 1:1000 in 4% skimmed milk), β-Actin (sc-47778, Santa Cruz, diluted 1:1000 in 4% skimmed milk), phospho Akt (S473, #4060, Cell Signaling, diluted 1:1000 in 4% BSA), and total Akt (#9272, Cell Signaling, diluted 1:1000 in 4% BSA), secondary horseradish-conjugated antibodies (goat anti-rabbit (#31460, Pierce) and goat anti-mouse (#31440, Pierce), diluted 1:1000-1:5000 in 4% skimmed milk) were added as appropriate and a chemiluminescent substrate kit (Thermo) used for detection.
Chrebp nb400 135
Manufactured by Novus Biologicals
Sourced in United States
ChREBP (NB400-135) is a recombinant protein produced in E. coli that represents the carbohydrate-responsive element-binding protein (ChREBP), a transcription factor that regulates genes involved in glucose and lipid metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Rabbit anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pparγ sc 7196, by Santa Cruz Biotechnology (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
4 protocols using chrebp nb400 135
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Nuclear Receptor Proteins
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Proteins were separated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA) and blotted onto PVDF membrane (MerckMillipore, Darmstadt, Germany). Primary antibodies used were LXRα (PP-K8607, R&D systems; 1:1000), LXRα LBD (PP-PPZ0412-00, R&D systems, Minneapolis, MN, USA; 1:1000), LXRβ (PP-K8917-00, R&D systems, Minneapolis, MN, USA; 1:1000), RXRα (sc-553, Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000), ChREBP (NB400-135, Novus Biologicals, Littleton, CO, USA; 1:1000), FLAG (F1804; Sigma-Aldrich, St. Louis, MO, USA; 1:1000) and β-actin (A5441; Sigma-Aldrich, St. Louis, MO, USA; 1:1000). Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG or mouse anti-rabbit IgG (115-035-174 and 211-032-171, Jackson ImmunoResearch Laboratories, West Grove, PA, USA; both 1:10,000).
3
Robust Molecular Signaling Pathway Analysis
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AS160 (07-741) was purchased from Millipore. PPARγ (sc-7196) and p70 S6K (sc-9027) are from Santa Cruz. ChREBP (NB400-135) is from Novus Biologicals. All other antibodies including Rictor (2140), Raptor (2280), HSL (4107), ATGL (2439), PRAS40 (2691), AKT (9272), GSK3β (9315), ACC (3676), ACLY (4332), FASN (3180), IR (3025), S473-AKT (4058), T308-AKT (4056), T24-FoxO1 (9464), T389-S6K (234), p-IR (3024), T246-pPRAS40 (2997), S660-pHSL (4126) and T642-pAS160 (4288), were purchased from Cell Signaling Technologies. 4-hydroxy-tamoxifen (4-OHT) was obtained from Toronto Research Chemicals. Rapamycin was purchased from LC Laboratories. Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), Tamoxifen, and all other reagents were from Sigma-Aldrich.
4
ChIP and DNase-seq Experiments
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ChIP experiments were performed according to standard protocol as described (Siersbaek et al., 2012) . Antibodies used were ChREBP (NB400-135, Novus Biologicals) and MED1 (M-255, sc-8998, Santa Cruz). DNase-Seq DNase-seq was performed on $10 million nuclei essentially as previously described (Siersbaek et al., 2011) .
Library Construction and Sequencing RNA-, DNase-, and ChIP-seq libraries were constructed using PentAdapters (Pentabase) essentially as described in Nielsen and Mandrup (2014) . Sequencing was performed on the Illumina HiSeq 1500 platform.
Library Construction and Sequencing RNA-, DNase-, and ChIP-seq libraries were constructed using PentAdapters (Pentabase) essentially as described in Nielsen and Mandrup (2014) . Sequencing was performed on the Illumina HiSeq 1500 platform.
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