Femur shaft nuclear protein extracts were isolated by homogenization in fractionation buffer (20 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA) containing protease inhibitor cocktail (Thermo-Fisher) following a brief centrifugation to separate the marrow. Homogenates were passed through an 18g needle and spun at 750 × g for 5 minutes. Nuclear pellets were resuspended in 1x TBS with 0.1% SDS and centrifuged again at 500 × g for 1 minute to remove any remaining insoluble tissue. Whole homogenates from the MSC cultures were solubilized in RIPA buffer (Thermo-Fisher). Protein extracts were analyzed for expression of total β-catenin using the Bolt Western Blot system (Thermo-Fisher) and associated reagents, using a goat polyclonal antibody (Cell Signaling #8814, Danvers, MA, USA). Results were normalized to TATA-binding protein (Abcam, Inc., Cambridge, MA #51841).
Tata binding protein
Manufactured by Abcam
Sourced in United States
TATA-binding protein (TBP) is a core transcription factor that plays a critical role in the initiation of transcription by RNA polymerase II. It binds to the TATA box, a DNA sequence element found in the promoter region of many genes, and helps recruit and position the RNA polymerase II complex to initiate transcription.
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Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Phospho c jun, by Cell Signaling Technology (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Adiponectin, by Abcam (1 mentions)
Citrate synthase, by Abcam (1 mentions)
γ tubulin, by Merck Group (1 mentions)
Normal rabbit igg, by Thermo Fisher Scientific (1 mentions)
H3k4me1, by Abcam (1 mentions)
Grhl2, by Merck Group (1 mentions)
H3k27ac, by Abcam (1 mentions)
Transforming growth factor β1, by Santa Cruz Biotechnology (1 mentions)
Cyp27a1, by Abcam (1 mentions)
16 protocols using tata binding protein
1
Fractionation and Analysis of Femur Shaft Nuclear Proteins
2
Immunoblotting of Nuclear and Cytosolic Proteins
3
Western Blot Analysis of Mitochondrial Proteins
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Proteins were resolved using NuPAGE Novex 4–12% Bis-Tris gels electroblotted onto PVDF membranes using the iBlot system (Invitrogen). Antibodies specific to the following proteins were used: Citrate synthase (Abcam Cat# ab129095 RRID:AB_11143209 ), MFN1 (Mitofusin-1 (D6E2S), Cell Signaling Techonology), MFN2 (Mitofusin-2 (D2D10), Cell Signaling Technology), Total Human OXPHOS cocktail (Mitosciences, Abcam), γ-tubulin (Sigma-Aldrich Cat# T5326 RRID:AB_532292 ), TATA binding protein (Abcam Cat# ab818 RRID:AB_306337 ), β-actin (Sigma-Aldrich Cat# A5316 RRID:AB_476743 ), PLIN1 (GP33, PROGEN Biotechnik), PLIN2 (GP47, PROGEN Biotechnik), adiponectin (Abcam Cat# ab13881 RRID:AB_2221613 ).
4
Antibody Validation for ChIP-seq and Western
5
Liver Protein Expression Analysis
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Frozen liver tissues were homogenized in buffer containing 0.25-M sucrose and 10-mM phosphate (pH 7.4). Nuclear fractions were extracted from parts of frozen liver samples using CelLyticTM NuCLEARTM Extraction Kits (Sigma-Aldrich Japan, Tokyo, Japan). Samples were then subjected to 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to polyvinylidene difluoride membranes as described previously [23 (link)]. Membranes were then incubated overnight at 4°C with primary polyclonal antibodies against alpha smooth muscle actin (α-SMA), cytochrome (CYP)7A1, CYP27A1 (Abcam plc, Cambridge, UK), constitutive androstane receptor (CAR; GeneTex, Inc., Irvine, CA), bile salt export pump (BSEP), CYP7B1, CYP8B1, farnesoid X receptor (FXR), pregnane X receptor (PXR), small heterodimer partner (SHP), sulfotransferase (SULT)2A1, transforming growth factor (TGF)-β1 (Santa Cruz Biotechnology, Santa Cruz, CA), or multidrug resistance-associated protein 3 (MRP3; Sigma-Aldrich Japan). GAPDH (Santa Cruz Biotechnology) and TATA binding protein (TBP; Abcam plc) were used as loading controls for homogenates and nuclear fractions, respectively. Protein signals were detected using ECL Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).
6
Western Blot Analysis of Renal Proteins
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Nuclear and cytoplasmic proteins from renal cortex tissues or cells were extracted with a commercial nuclear extraction kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA, USA). Protein concentration was determined with the bicinchoninic acid method (Pierce Pharmaceuticals, Rockford, IL, USA). For western blotting, 40 μg of protein was electrophoresed on a 10% SDS-PAGE minigel. The proteins were transferred onto polyvinylidene difluoride membrane, which was hybridized in blocking buffer overnight at 4 °C with goat polyclonal anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit polyclonal anti-NF-kB p65 antibody (1:1000, Cell Signaling Technology, USA), rabbit monoclonal anti-phospho IκBα antibody (1:1000, Cell Signaling Technology), rabbit polyclonal anti-Nox1 (1:1000, Abcam Plc, Cambridge, MA, USA), rabbit polyclonal anti-Nox2 (1:1000, Bioworld Technology, St Louis Park, MN, USA), rabbit polyclonal anti-Nox4 (1:1000, Bioworld Technology), mouse monoclonal anti-β actin antibody (1:5000, Sigma-Aldrich) or TATA binding protein (1:2000, Abcam Plc). The membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibody diluted 1:1000 for 60 min at room temperature. The signals were detected with the enhanced chemiluminescence method (Amersham, Buckinghamshire, UK).
7
Cytoplasmic and Nuclear Protein Extraction
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At 60 min after the third HBO treatment, the cells were rinsed with PBS, treated with 0.05% trypsin, and then collected by centrifugation. NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, USA) were used to isolate cytoplasmic and nuclear extracts from the cells. The proteins were separated by 10% SDS-PAGE. After blocking with 10% nonfat milk, the membranes were incubated overnight at 4 °C with a 1000-fold dilution of a mouse antibody against IκBα (Cell Signal, USA) or β-actin (Abcam, UK) for cytoplasmic extracts and NF-κB p65 (Cell Signal, USA) or the TATA binding protein (TBP; Abcam, UK) for nuclear extracts. The membranes were then washed and rinsed with ECL detection reagents. The band images were photographed using Hyperfilm. The intensity of the staining for IκBα, β-actin, NF-κB p65, and TATA was quantified using an image analysis system.
8
Regulation of Lipid Metabolism in Prostate and Lung Cancer Cells
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All the cell lines were from from ATCC (Manassas, VA). LNCaP, DU145, PC3 and A549 cells were cultured in RPMI1640 media with 10% FBS (fetal bovine serum). PWR-1E, RWPE-1, WPE-NA22 and WPE-NB14 were maintained in keratinocyte growth medium with 5 ng/mL human recombinant epidermal growth factor and 0.05 mg/mL bovine pituitary extract; and as per experimental requirements, these cells were also cultured with 10% FBS or serum free keratinocyte growth medium. Media, serum and other cell culture materials were from Invitrogen-Life Technologies (Carlsbad, CA). Silibinin and fatostatin were from Sigma (St Louis, MO) and dissolved in DMSO as stock solution. The final concentration of DMSO in the culture medium during different treatments did not exceed 0.1% (v/v). Antibodies for SREBP1, HMGCR, and TATA-binding protein (TBP) were obtained from Abcam (Cambridge, MA). Total and/or phosphorylated antibodies for SREBP1, SREBP2, FASN, ACC, ACYL, AMPK, and AMACR were from Cell Signaling Technologies (Danvers, MA). Tubulin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Compound C was from Calbiochem (La Jolla, CA). R1881 was from Perkin Elmer (Waltham, MA). ECL detection system and anti-mouse HRP-conjugated secondary antibody were from GE Healthcare (Buckinghamshire, UK).
9
Molecular Signaling Pathway Analysis
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Cells were lysed in RIPA buffer protease inhibitor mixture Complete Mini (Roche) and total protein concentration was quantified by Bradford assay (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). Samples were probed with antibodies against RET (C-20), p-RET (Tyr1062), Notch1 (C-20), HES1 (H-140), Deltex1 (D-20) from Santa Cruz, cleaved Notch1 (Val1744) (Cell Signaling), NF-κB p65 and NF-κB p65 (pS536) from BD Biosciences, TATA-binding protein (TBP; Abcam), and β-Actin (Sigma).
For immunofluorescence, cells were grown on glass slides for 24 h and fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 10% FCS and 1% BSA in PBS. Cells were incubated with primary antibody for NOTCH1, HES1, Deltex1 and NF-κB p65 (Santa Cruz) diluted in 3% BSA in PBS/0.02% Triton X-100, washed in PBS, and incubated for 60 min with fluorescence-labelled secondary antibody (Thermo Scientific). Cell nuclei were stained with DAPI solution. Images were obtained using an inverted confocal laser scanning microscope (Zeiss).
For immunofluorescence, cells were grown on glass slides for 24 h and fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 10% FCS and 1% BSA in PBS. Cells were incubated with primary antibody for NOTCH1, HES1, Deltex1 and NF-κB p65 (Santa Cruz) diluted in 3% BSA in PBS/0.02% Triton X-100, washed in PBS, and incubated for 60 min with fluorescence-labelled secondary antibody (Thermo Scientific). Cell nuclei were stained with DAPI solution. Images were obtained using an inverted confocal laser scanning microscope (Zeiss).
10
Subcellular Fractionation and Western Blot Analysis
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Cells were lysed with RIPA buffer. Nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic extraction reagents (Pierce). Protein concentrations were determined using the DCTM Protein Assay (Bio-Rad). Following SDS-PAGE, Western Blot analysis was performed using the following primary antibodies: β-catenin (Millipore), NFAT1 (Cell Signaling), pan-Akt (Cell Signaling), phospho-Akt (Ser473, Cell Signaling), TATA-binding protein (TBP, Abcam), and α-tubulin (Developmental Studies Hybridoma Bank). A horseradish peroxidase-conjugated antibody (Millipore) was used as a secondary antibody, and Pierece ECL Western Detection kit (ThermoFisher) was used for protein visualization.
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