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GABPα is a protein that functions as a transcription factor, regulating the expression of various genes. It plays a role in cellular processes such as cell growth and differentiation. GABPα is a commonly used protein in biological research and laboratory applications.

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Lab products found in correlation

Hiseq 2000, by Illumina (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions) Cyclin d2, by Cell Signaling Technology (1 mentions) Facs sorting, by BD (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions) Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions) Control igg, by Cell Signaling Technology (1 mentions) 7900ht fast real time system, by Thermo Fisher Scientific (1 mentions) Super block, by ScyTek Laboratories (1 mentions) Permount mounting medium, by Thermo Fisher Scientific (1 mentions) Dab peroxidase substrate kit, by Vector Laboratories (1 mentions) Tom20, by Cell Signaling Technology (1 mentions) Chemidoc mp imager, by Bio-Rad (1 mentions) C ebpε, by Santa Cruz Biotechnology (1 mentions) Image lab software, by Bio-Rad (1 mentions) Hrp conjugated secondary ab, by Merck Group (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions)

5 protocols using gabpα

1

ChIP-Seq Analysis of Transcription Factors

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ChIP was performed as previously described (22 (link),36 (link),37 (link)) using the following antibodies: AR N20 [SC-816X, Santa Cruz], GABPα [SC-22810, Santa Cruz], ERG [SC-353, Santa Cruz]. Biological replicates were used. Enrichment was tested with 6 μl DNA by real-time PCR using SYBRgreen (Applied Biosystems). Single-end SOLEXA libraries were prepared as previously described (36 (link)) and 36 bp sequence reads were generated by the Illumina HiSeq 2000. Sequence reads were aligned against the Human Reference Genome (assemby hg18, NCBI Build 36 (link)) using Burrows-Wheeler Aligner (BWA) version 0.5.5 (38 (link)). Reads were filtered by removing those with a BWA alignment quality score less than 15 as well as duplicate reads. Enriched regions of the genome were identified by comparing ChIP samples to input samples using MACS (39 (link)) and SWEMBL (http://www.ebi.ac.uk/∼swilder/SWEMBL/). Only peaks that were identified by both MACS and SWEMBL (high confidence peaks) were used for further analyses.
Sharma N.L., Massie C.E., Butter F., Mann M., Bon H., Ramos-Montoya A., Menon S., Stark R., Lamb A.D., Scott H.E., Warren A.Y., Neal D.E, & Mills I.G. (2014). The ETS family member GABPα modulates androgen receptor signalling and mediates an aggressive phenotype in prostate cancer. Nucleic Acids Research, 42(10), 6256-6269.
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2

T Cell Activation Protein Profiling

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CD4+ and CD8+ T cells were purified from spleen and lymph nodes of CD4CreGabpaf/f mice and littermate controls by FACS sorting (BD, Aria). In the time course culture experiment, naive T cells were purified, subject to α-CD3/28 and IL-2 culture, and collected at indicated time points. Total protein extracts were dissolved in SDS sample buffer, separated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membrane (Millipore). The membranes were probed with antibodies against GABPα (sc-22810, Santa Cruz), CDK6 (3136, Cell Signaling), Cyclin D2 (3741, Cell Signaling), Mcm3 (4003, Cell Signaling), Mcm5 (A300-195A, Bethyl Laboratories), GADPH (ab9485, Abcam), and β-actin (AC-15, Sigma), and visualized with the Immobilon Western Chemiluminescent HRP Substrate (Millipore).
Luo C.T., Osmanbeyoglu H.U., Do M.H., Bivona M.R., Toure A., Kang D., Xie Y., Leslie C.S, & Li M.O. (2017). Ets transcription factor GABP controls T cell homeostasis and immunity. Nature Communications, 8, 1062.
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3

ChIP-qPCR Analysis of GABPA Binding

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Chromatin immunoprecipitation (ChIP) for GABPα was performed using the ActiveMotif High Sensitivity kit. In brief, GBM1, T98G, HCT116, and HEK293T CRISPR controls and β1L-reduced clones were grown to 80% confluency in 15 cm plates and fixed with 4% formaldehyde. Chromatin was sonicated to a size range of 200–1200 bp by the Diagenode Biorupter. 12–18 μg of chromatin was used per GABPα (Santa Cruz Biotechnology: sc-22810) and IgG control (Cell Signaling: 2729) immunoprecipitation for each cell type. Enrichment at the TERT promoter was determined by qPCR with the ssoAdvanced Universal SYBR Green Supermix (Biorad) supplemented with Resolution Solution from GC-RICH PCR System (Roche). The following primer set was used for qPCR: TERT+47 (forward: 5’-GCCGGGGCCAGGGCTTCCCA-3’; reverse: 5’ CCGCGCTTCCCACGTGGCGG-3’; Tm=74° Celsius). PCR was carried out on the Applied Biosystems 7900HT Fast Real-Time System. Three replicate PCR reactions were carried out for each sample.
Mancini A., Xavier-Magalhães A., Woods W.S., Nguyen K.T., Amen A.M., Hayes J.L., Fellmann C., Gapinske M., McKinney A.M., Hong C., Jones L.E., Walsh K.M., Bell R.J., Doudna J.A., Costa B.M., Song J.S., Perez-Pinera P, & Costello J.F. (2018). Disruption of the β1L isoform of GABP reverses glioblastoma replicative immortality in a TERT promoter mutation-dependent manner. Cancer cell, 34(3), 513-528.e8.
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4

Immunohistochemical Analysis of Adipose Tissue

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Freshly isolated iWAT was fixed in 10% PBS-buffered formalin, dehydrated, and embedded in paraffin for sectioning. Five-micron sections were de-paraffinized, and the antigen was retrieved using 10 mM sodium citrate buffer containing 0.05% Tween20, pH 6.0. After blocking the endogenous peroxidases and non-binding sites with superblock (ScyTek Laboratories Inc., Logan, UT), the sections were incubated with primary antibodies (UCP1, 1:250; TOM20, 1:250, Cell Signaling Technology, and GABPα, 1:100, Santa Cruz Biotechnology) overnight at 4°C. After several washes, the slides were incubated with Dky Rabbit IgG Biotin secondary antibody (1:500, EMD Millipore, Burlington, MA) and developed using the DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA). The color development time was optimized by monitoring the signal under a microscope. The nuclei were stained with hematoxylin, the slides were mounted using a Permount mounting medium (Fischer Scientific), and the images were captured using an EVOS microscope.
Mooli R.G., Zhu B., Khan S.R., Nagati V., Michealraj K.A., Jurczak M.J, & Ramakrishnan S.K. (2024). Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis. Frontiers in Endocrinology, 15, 1385811.
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5

Western Blot Analysis of Protein Extracts

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For all Western blot analyses, 1 × 107 cells were collected, washed with PBS supplemented with protease inhibitor cocktail (11836153001, Sigma-Aldrich) and 1 mM PMSF (Sigma-Aldrich), and then lysed with Laemmli lysis buffer as previously described (46 (link)). Equal amounts of the protein extracts (~10 μg) were electrophoresed on NuPAGE 4–12% Bis-Tris gradient gels (Novex, Carlsbad, CA) and then electro-blotted onto Immobilon PVDF membranes (EMD Millipore, Billerica, MA). Membranes were probed with the following Abs and dilutions: GABPα (1:1000; SC-22810), PU.1 (1:1000; SC-352), C/EBPε (1:1000; SC-158), and β-actin (1:1000) (all from Santa Cruz Biotechnology, Dallas, TX), LBR from guinea pig serum [kindly provided by Prof. Harald Herrmann, German Cancer Research Institute, Heidelberg, Germany and previously described (20 (link))], or tubulin (1:1500; Sigma-Aldrich). HRP-conjugated secondary Ab (1:2000, Sigma-Aldrich) was used against primary Abs, and chemiluminescence was detected using Immobilon Western HRP substrate solution (EMD Millipore). Images were captured from exposed and processed film or with the ChemiDocMP Imager using Image Lab Software (BioRad Laboratories).
Malu K., Garhwal R., Pelletier M.G., Gotur D., Halene S., Zwerger M., Yang Z.F., Rosmarin A.G, & Gaines P. (2016). Cooperative activity of GABP with PU.1 or C/EBPε regulates lamin B receptor gene expression, implicating their roles in granulocyte nuclear maturation. Journal of immunology (Baltimore, Md. : 1950), 197(3), 910-922.
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