Gata1

Manufactured by Abcam

Sourced in United States

GATA1 is a transcription factor that plays a crucial role in the regulation of genes involved in erythroid and megakaryocytic cell differentiation. It acts as a master regulator of the hematopoietic system.

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8 protocols using gata1

Western Blot Analysis of Key Proteins

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Cells were lysed in Laemmli sample buffer (BioRad, Cat# 161-0737) and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane and incubated with antibodies to PTBP1(1:1000, Abcam, Cat# ab5642), GAPDH (1:5000, Cell Signaling Technology, Cat#5174), ALAS2 (1:1000, Abcam, Cat# ab184964) or GATA1 (1:1000, Abcam, Cat# ab11852). Horseradish peroxidase-conjugated secondary antibodies were used. Signals were detected using an ECL kit (Invitrogen, Cat# WP20005). Unprocessed images of all western blots are shown in Supplementary Fig. 8.

Liu J., Li Y., Tong J., Gao J., Guo Q., Zhang L., Wang B., Zhao H., Wang H., Jiang E., Kurita R., Nakamura Y., Tanabe O., Engel J.D., Bresnick E.H., Zhou J, & Shi L. (2018). Long non-coding RNA-dependent mechanism to regulate heme biosynthesis and erythrocyte development. Nature Communications, 9, 4386.

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Western Blot Analysis of Mitochondrial and Epigenetic Proteins

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Western blot was performed as described^{2 (link)} using the following antibodies: TFAM (7495S, Cell Signaling Technology; sc-23588, Santa Cruz Technology), PHB2 (14085S, Cell Signaling Technology), ATP5A1 (sc-136178, Santa Cruz Technology), ATP5B (sc-16690, Santa Cruz Technology), ATP5D (ab97491, Abcam), ATP5O (ab110276, Abcam, clone 4C11C10D12), 4EBP1 (9644S, Cell Signaling Technology), phos-4EBP1 (Thr37/46) (2855S, Cell Signaling Technology), p70S6K (2708S, Cell Signaling Technology), phos-S6K (Ser235/236) (2211S, Cell Signaling Technology), GATA1 (ab47490, Abcam), GATA2 (ab22849, Abcam), TSC1 (6935, Cell Signaling Technology), TSC2 (4308, Cell Signaling Technology), H3K9ac (9649, Cell Signaling Technology), H3K27ac (8173, Cell Signaling Technology), H3K56ac (4243, Cell Signaling Technology), H4K5ac (ab51997, Abcam), H3K4me3 (9571, Cell Signaling Technology), H3K9me3 (13969, Cell Signaling Technology), H3K27me3 (9733, Cell Signaling Technology), H3K36me3 (4909, Cell Signaling Technology), H3 (4499, Cell Signaling Technology), ACTB (MAB1501, Millipore, clone C4), and GAPDH (sc-26778, Santa Cruz Biotechnology). Densitometry quantification was performed using ImageJ software. All antibodies were used at 1:1,000 dilutions except phos-4EBP1 (1:500), phos-S6K (1:500) and ACTB (1:2,500).

Liu X., Zhang Y., Ni M., Cao H., Signer R.A., Li D., Li M., Gu Z., Hu Z., Dickerson K.E., Weinberg S.E., Chandel N.S., DeBerardinis R.J., Zhou F., Shao Z, & Xu J. (2017). Regulation of Mitochondrial Biogenesis in Erythropoiesis by mTORC1-Mediated Protein Translation. Nature cell biology, 19(6), 626-638.

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Chromatin Immunoprecipitation by CUT&RUN

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CUT&RUN was performed using the CUT&RUN Kit from Cell Signaling (catalog no. 86652S) according to the manufacturer’s instructions. Briefly, 0.2–0.8 million cells were harvested and washed twice in 1× wash buffer at room temperature and bound to 15 μl of Concanavalin A beads. Primary antibody incubation was performed in PCR tubes for 2 h at 4 °C with rotation. The following antibodies were used: H3K27ac (Invitrogen, catalog no. MA5–23516), GATA1 (Abcam, catalog no. ab11852). After two washes, pAG-MNase was added and incubated for 1 h at 4 °C with rotation. After three washes, cells were resuspended in 1× wash buffer and pAG-MNase digestion was activated by adding 2 mM CaCl₂. Samples were incubated on ice for 45 min, then 2× stop buffer was added to end the reaction. The samples were incubated at 37 °C for 10 min to release the captured chromatin fragments. The sequencing libraries were prepared using the NEBNext Ultra II DNA Library (New England Biolabs, catalog no. E7645) with 15–30 ng of input DNA. Sequencing libraries were pooled and pair-end (2 × 50 bp) sequenced on an Illumina Nextseq 2000 platform.
Reads were aligned to the reference genome using Bowtie1 with parameters --best --sam --chunkmbs 256 -X 800. Peaks were called using MACS2. Peaks overlapped with the blacklist were further filtered out using bedtools.

Huang P., Peslak S.A., Ren R., Khandros E., Qin K., Keller C.A., Giardine B., Bell H.W., Lan X., Sharma M., Horton J.R., Abdulmalik O., Chou S.T., Shi J., Crossley M., Hardison R.C., Cheng X, & Blobel G.A. (2022). HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription. Nature genetics, 54(9), 1417-1426.

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CUT&RUN Profiling for Transcription Factors

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CUT&RUN experiments were carried out as described (Skene and Henikoff, 2017 ) with modifications. Briefly, nuclei from 2×10⁶ cells were isolated with NE buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol and 1× protease inhibitor cocktails from Sigma), captured with BioMag®Plus Concanavalin A (Polysciences) and incubated with primary antibody for 2 hours. After washing away unbound antibody with wash buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1× protease inhibitor cocktails from Sigma), protein A-MNase was added at a 1:1000 ratio and incubated for 1 hour. The nuclei were washed again and placed in a 0°C metal block. To activate protein A-MNase, CaCl₂ was added to a final concentration of 2 mM. The reaction was carried out for different time courses and stopped by addition of equal volume of 2×STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A and 40 μg/mL glycogen). The protein-DNA complex was released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit fluorometer and bioanalyzer quality control. Protein A-MNase (batch 5) was kindly provided by Dr. Steve Henikoff. The antibodies used were: GATA1, ab11852, abcam; CTCF, 07-729, Millipore; BCL11A, ab191401, abcam; normal rabbit IgG, 12-370, Millipore.

Liu N., Hargreaves V.V., Zhu Q., Kurland J.V., Hong J., Kim W., Sher F., Macias-Trevino C., Rogers J.M., Kurita R., Nakamura Y., Yuan G.C., Bauer D.E., Xu J., Bulyk M.L, & Orkin S.H. (2018). Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. Cell, 173(2), 430-442.e17.

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Whole-Cell Protein Extraction and Western Blotting

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The whole-cell extracts were prepared by washing cells twice with ice-cold 1× PBS and lysed in lysis buffer with the addition of Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktails and PMSF (Kangchen bio-tech, China) for 30 min at 4 °C on a rocking platform before scraping and transferring to tubes. The lysates were cleared by centrifugation at 13,000 g for 20 min at 4 °C, and protein quantification was performed with the BCA protein assay kit (Beyotime, China). For western blotting, 20 μg total protein were prepared with 5× SDS sample buffer and resolved by 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane (ImmobilinP, Millipore) using standard procedures. After blocking in 5% skim milk in Tris-buffered saline–Tween (TBST), incubation for overnight at 4 °C with primary antibodies against GAPDH, β-Tubulin, Sp1, Sp3 (Santa Cruz, USA), GATA1 and STING (Abcam, UK) and then with HRP-conjugated secondary antibodies (Abcam, UK) was performed. Subsequently, Chemoluminescense signals of membranes were quantified with ECL reagent (Pierce, USA) and visualized by enhanced chemiluminescence (Cell Signaling Technology, Inc). Anti-GAPDH or β-Tubulin was used as loading control.

Xu Y.Y., Jin R., Zhou G.P, & Xu H.G. (2017). Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells. Scientific Reports, 7, 2090.

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Western Blot Analysis of Mitochondrial and Epigenetic Proteins

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Western Blotting Techniques for Protein Analysis

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Western blotting reagents were purchased from Thermo Fisher Scientific unless otherwise stated. Cells were lysed in M-Per mammalian protein extraction reagent (cat#78501) supplemented with Halt® protease and phosphatase inhibitor cocktail, following manufacturer’s instructions. Quantification of total protein was achieved by using a bicinchoninic acid (BCA) assay. For each sample, 20–40μg of protein were separated by electrophoresis using 12% Tris-Glycine gels. Protein transfers were performed using the iBlot2 system (20V for 1 minute, 23V for 3 minutes, and 25V for 2 minutes setting). PVDF membranes were blocked in 4% milk- Tris Buffered Saline with Tween 20 (TBST) buffer for 1 h, incubated in primary antibody overnight followed by the HRP secondary for 1 h.
The sources of primary antibodies used were: GATA-1 (Abcam; cat#89505, mouse monoclonal), acetylated tubulin (Santa Cruz, Dallas, TX; cat#sc-23950, mouse monoclonal), alpha tubulin (Abcam; cat#4074, rabbit polyclonal), histone H3 (ProSci, Poway, CA; cat#31007), and acetylated histone H3 (Thermo Fisher Scientific; Lys6, cat#MA5–11195 and Lys36, cat#MA-24672). Secondary HRP labeled, anti-mouse (cat#6728) and anti-rabbit (cat#6721) antibodies were purchase from Abcam. Membranes were incubated for 1 min in ECL reagent and captured using ChemiDoc MP (BioRad, Hercules, CA).

Simic D, & Sang N. (2019). Compounds targeting Class II histone deacetylases do not cause panHDACi associated impairment of megakaryocyte differentiation. Experimental hematology, 72, 36-46.

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ChIP-seq protocol for histone marks and transcription factors

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Chromatin immunoprecipitation (ChIP) was performed on 3–5 × 10⁶ cells per replicate using the ChIP Assay Kit (Cat. No. 17-295, Millipore). All stages beyond the fixation stage used kit solutions as per the manufacturer’s instructions. A single 10 min 1% formaldehyde fixation was used for all antibodies. The following antibodies and amounts/dilutions were used: CTCF 1:100 (Cat. No. 07-729, Millipore), H3K27ac 1:1000 (Cat. No. ab4729, AbCam), H3K4me1 1:5000 (Cat. No. 07-436, Merck), H3K4me3 1:200 (Cat. No. ab8580, AbCam), GATA1 1:200 (Cat. No. ab11852, AbCam), H2AK119ub 1:100 (Cat No. #8240, CST) H3K27me3 1:100 (Cat. No. 9733, Cell Signaling Technologies). The cross-linking reaction was quenched by the addition of glycine (Sigma-Aldrich) to a final concentration of 130 mM), then washed once in PBS and snap-frozen. Samples were stored at −80 °C prior to further processing. Chromatin fragmentation was performed using the Covaris S220 Focused ultra-sonicator with the following settings (Duty Cycle: 2%, Intensity: 3.0, Cycles/burst: 200, Power mode: Freq. Sweeping, Duration: 120 s, Temp: 6 °C, Batches: 4) to obtain an average fragment size between 200 bp and 500 bp. DNA libraries for sequencing were prepared with the NEBNext Ultra II DNA library prep kit (New England Biolabs) and sequenced on the Illumina platform.

King A.J., Songdej D., Downes D.J., Beagrie R.A., Liu S., Buckley M., Hua P., Suciu M.C., Marieke Oudelaar A., Hanssen L.L., Jeziorska D., Roberts N., Carpenter S.J., Francis H., Telenius J., Olijnik A.A., Sharpe J.A., Sloane-Stanley J., Eglinton J., Kassouf M.T., Orkin S.H., Pennacchio L.A., Davies J.O., Hughes J.R., Higgs D.R, & Babbs C. (2021). Reactivation of a developmentally silenced embryonic globin gene. Nature Communications, 12, 4439.

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