Gata1

Manufactured by Santa Cruz Biotechnology

Sourced in Netherlands

GATA1 is a transcription factor that plays a crucial role in the development and differentiation of blood cells, particularly erythroid cells. It regulates the expression of genes involved in hematopoiesis and erythropoiesis. GATA1 is an important component in the regulation of gene expression during blood cell development.

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8 protocols using gata1

Immunoprecipitation and Western Blot Protocol

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Briefly, 10 × 10⁶ cells were collected and lysed in 1 ml of RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for immunoprecipitation. Cell extracts were pre-cleared by mixing lysates with 2 μg of normal control IgG and 50 μl of protein G agarose and incubating for 4 h at 4 °C. The pre-cleared lysates were mixed with 2 μg of the appropriate antibodies plus 20 μl of protein G agarose beads and rotated overnight at 4 °C. The immunoprecipitated complexes were obtained by centrifugation, washed three times with RIPA lysis buffer and boiled at 75 °C in loading buffer for 10 min for western blot. Cell lysates were also directly used for western blot analysis. All western blot photos were representative photos from at least two experiments with similar results. Antibodies for HSC 70 (cat no. SC-7298), Lyn (cat no. SC-7274), Fyn (cat no. SC-16), PSTPIP2 (cat no. SC-83015), GATA1 (cat no. SC-1234), SHC (cat no. SC-1695) were purchased from Santa Cruz Biotechnology. Antibody for FLAG (cat no. F3165) was obtained from Sigma. Antibodies for ERK (cat no. 4695), p-ERK (cat no. 4370), p-SRC (cat no. 2101) were purchased from Cell Signaling Technology.

Liu L., Wen Q., Gong R., Gilles L., Stankiewicz M.J., Li W., Guo M., Li L., Sun X., Li W., Crispino J.D, & Huang Z. (2014). PSTPIP2 dysregulation contributes to aberrant terminal differentiation in GATA-1-deficient megakaryocytes by activating LYN. Cell Death & Disease, 5(1), e988-.

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Erythroid differentiation regulation

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Desmethylimipramine (desipramine), N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamide dihydrochloride (GW4869), SMase from Staphylococcus aureus, C2 dihydroceramide, S1P, and BafA1 were purchased from Sigma-Aldrich (Bornem, Belgium), C2-ceramide from Santa Cruz Biotechnology (Boechout, Belgium) and C6-ceramide from Enzo Life Sciences (Antwerp, Belgium). Human recombinant TNFα was kindly provided by Pr. Athanase Visvikis (Université de Lorraine, Nancy, France). Stem cell factor (SCF) and interleukin (IL)-3 were purchased from ReliaTech (Wolfenbüttel, Germany). The erythroid differentiation is induced with human recombinant erythropoietin (Epoietin beta, Neorecormon Roche, Grenzach-Whylen, Germany). The primary antibodies used were directed against: GATA-1, GATA-2, PU.1, α-, β-, and γ-globin (Santa Cruz Biotechnology), SphK1, PI3K, P-PI3K^Y458, AKT, P-AKT^S473, P-ULK1^S758, mTOR, P-mTOR^S2448, Atg7, Atg5-Atg12, Atg13, P-Atg13^S355, Beclin-1, p62/SQSTM1 (Cell Signaling, Leiden, The Netherlands), LC3 (Sigma), Rubicon (Abcam, Cambridge, UK), and ceramide MID 15B4 (Enzo Life Sciences, Antwerpen, Belgium), and the antibodies used for flow cytometry (CD235a/GPA, CD11b) were purchased from BD BioSciences (Erembodegem, Belgium).

Orsini M., Chateauvieux S., Rhim J., Gaigneaux A., Cheillan D., Christov C., Dicato M., Morceau F, & Diederich M. (2018). Sphingolipid-mediated inflammatory signaling leading to autophagy inhibition converts erythropoiesis to myelopoiesis in human hematopoietic stem/progenitor cells. Cell Death and Differentiation, 26(9), 1796-1812.

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Testicular Immunohistochemistry Protocol

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Immunohistochemistry analysis of testicular sections was performed using 4% paraformaldehyde fixed tissues as previously described^{65 (link)}. Primary antibodies were diluted at 1:50 (GATA1; Santa Cruz Biotechnology sc265), 1:500 (PLZF; R&D Systems AF2944), 1:500 (SYCP3; Abcam ab97672), and 1:200 (STAR; CST 8449 and αSMA; Sigma A2547). Biotinylated polyclonal goat anti-rabbit (Dako, E0432) and anti-mouse (Dako, E0433) antibodies were diluted 1:500. Histological sections were examined using an AxioImager microscope equipped with an AxioCam ICc1 camera and ZEN v.2.3 (Blue edition, Zeiss).

Jamin S.P., Petit F.G., Kervarrec C., Smagulova F., Illner D., Scherthan H, & Primig M. (2017). EXOSC10/Rrp6 is post-translationally regulated in male germ cells and controls the onset of spermatogenesis. Scientific Reports, 7, 15065.

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Chromatin Immunoprecipitation (ChIP) Assay

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Chromatin immunoprecipitation (ChIP) experiments were performed^{40 (link)}. In all, 7 × 10⁷−1 × 10⁸ cells were used for each ChIP. Cells were crosslinked with 1% formaldehyde for 10 min and the reaction was quenched by addition of 2.5 mM glycine. Fixed cells were sonicated at high voltage for 20 min (30 s on, 30 seconds off) using Bioruptor® (Diagenode) to obtain 200–300 bp DNA fragments. The fragmented chromatin was pulled down at 4 °C overnight using an antibody against GATA-1 (Santa Cruz Biotechnology, #sc-256, 1:5000 dilution). Chromatin cross-linking was then reversed at 65 °C overnight followed by DNA purification. Real-time qPCR was performed on ChIP DNA on an Applied Biosystems ViiA7 Real-time PCR System (Applied Biosystems). Primers used for ChIP-qPCR are listed in Supplementary Table 5.

Yang L., Chen Z., Stout E.S., Delerue F., Ittner L.M., Wilkins M.R., Quinlan K.G, & Crossley M. (2020). Methylation of a CGATA element inhibits binding and regulation by GATA-1. Nature Communications, 11, 2560.

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Antibody Preparation for Cellular Analysis

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Antibodies against histone H1, Hsc70, lamin B, and GATA1 were purchased
from Santa Cruz Biotechnology. Antibodies against histone H3, caspase-3, and
Ezh2 were purchased from Cell Signaling Technology. Polyclonal antibodies
against other histones analyzed, GAPDH and HDAC2 were purchased from Abcam. All
the antibodies for flow cytometric analysis were purchased from eBioscience.
Alexa Fluor 488 and Alexa Fluor 594 for immunofluorescence stains were purchased
from Jackson ImmunoResearch. Nuclear export inhibitor leptomycin B was purchased
from Cell Signaling Technology. Caspase-3 inhibitor and Pan-caspase inhibitor
Z-VAD-FMK were purchased from Santa Cruz Biotechnology. Mimosine and aphidicolin
were purchased from Sigma.

Zhao B., Mei Y., Schipma M.J., Roth E.W., Bleher R., Rappoport J.Z., Wickrema A., Yang J, & Ji P. (2016). Nuclear condensation during mouse erythropoiesis requires caspase-3-mediated nuclear opening. Developmental cell, 36(5), 498-510.

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Western Blot Analysis of Hematopoietic Regulators

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Western blotting analysis was performed using NuPAGE precast gels (3–8% Tris-acetate for JARID1A and 4–12% Bis–tris for SCL, GATA1, LMO2 and LDB1; Life Technologies) according to the manufacturer's instructions. Primary antibodies used were: JARID1A (ab70892; Abcam), SCL (sc-12984; Santa Cruz), LDB1 (sc-11198; Santa Cruz), LMO2 (MCA2744GA, ABD serotec) and GATA1 (sc-1234; Santa Cruz). Secondary antibodies used were horseradish peroxidase conjugated anti-goat/mouse/rabbit immunoglobulin (Santa Cruz). Protein detection was carried out using ECL prime reagent kit (Amersham).

Karia D., Gilbert R.C., Biasutto A.J., Porcher C, & Mancini E.J. (2020). The histone H3K4 demethylase JARID1A directly interacts with haematopoietic transcription factor GATA1 in erythroid cells through its second PHD domain. Royal Society Open Science, 7(1), 191048.

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Quantitative Western Blot Analysis of Phosphorylated GATA1

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Cells were washed with PBS and lysed with ice cold IP Lysis/Wash Buffer (Pierce) containing protease inhibitor cocktail (Sigma) and Halt phosphatase inhibitor (Pierce), followed by centrifugation and storage at −80°C. A BCA Assay (Pierce) was performed to quantify protein concentrations. Proteins were reduced with 20 mM DTT, resolved on a NuPage 4–12% Tris-Bis gel (Invitrogen), transferred to a PVDF membrane, and blocked overnight in TBS-T containing 5% BSA. Membranes were subsequently blotted on a SnapID system (Millipore) using antibodies against p-GATA1 (Assay biotech), GATA1, or β-actin (Santa Cruz Biotechnology) (all diluted 3:1000), and species appropriate HRP-conjugated secondary antibodies (3:1000). HRP was detected with ECL-Plus (GE Healthcare) using a Typhoon imaging system. Blotting was quantified with densitometry using ImageJ. Sample bands were normalized to the corresponding β-actin band and then normalized to the average appropriate control samples. A student’s t-test with fdr adjustment was used to assess Western blot data.

Weiss M.S., Bernabé B.P., Shin S., Asztalos S., Dubbury S.J., Mui M.D., Bellis A.D., Bluver D., Tonetti D.A., Saez-Rodriguez J., Broadbelt L.J., Jeruss J.S, & Shea L.D. (2014). Dynamic transcription factor activity and networks during ErbB2 breast oncogenesis and targeted therapy. Integrative biology : quantitative biosciences from nano to macro, 6(12), 1170-1182.

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Immunohistochemical Analysis of RLIM Expression

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Primary antibodies used for immunostainings were rabbit RLIM (Ostendorff et al. 2002) , Lectin PNA from Arachis hypogaea (peanut), GATA1 (Santa Cruz Biotechnology, sc265), GATA4 (Abcam, ab84593), pPKAs (Cell Signaling, # 9624), PY (Millipore, clone G10), Alexa Fluor 488-conjugated lectin peanut agglutinin (ThermoFisher, # L21409).
Secondary antibodies were Alexa Fluor® 488 Donkey Anti-Rabbit IgG (Invitrogen, A21206), Alexa Fluor® 488 Goat Anti-mouse IgG (Invitrogen, A11029), Alexa Fluor® 568 Goat Anti-Rabbit IgG (Invitrogen, A11011), Alexa Fluor® 568 Goat Anti-rat IgG (Invitrogen, A11077), and Alexa Fluor® 568 Goat Anti-mouse IgG (Invitrogen, A11004).
Immunohistochemical (co-)staining on paraffin-embedded tissue sections were carried out as previously reported (Shin et al. 2010) . Northern blot analysis was performed on a membrane containing RNA from adult mouse tissues (Clontech). As probe, we used a 568 bp SacI fragment isolated from mouse Rlim cDNA, which was 32 P-labeled via random priming (Gibco BRL) as previously described (Ostendorff et al. 2000) .

Wang F., Gervasi M.G., Bošković A., Sun F., Rinaldi V.D., Yu J., Wallingford M.C., Tourzani D.A., Mager J., Zhu L.J., Rando O.J., Visconti P.E., Strittmatter L., & Bach I. (2020). Deficient spermiogenesis in mice lackingRlim.

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