Total proteins were extracted from cells or tissues using a RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF, 5 μg/ml aprotinin and 5 μg/ml leupeptin. The protein concentration was determined with a BCA Protein Assay Kit (Vigorous, China). The total protein (15–30 μg) was loaded onto a 10% SDS-PAGE gel, probed with mouse or rabbit mAb against MECOM (Epitomics, MA, USA), GATA2 (Proteintech, IL, USA), p-c-Jun (Bioworld Technology, St. Louis, USA), c-Jun (Bioworld Technology), PU.1 (Cell Signaling Technology) and GAPDH (Proteintech) followed by horseradish peroxidase-conjugated sheep anti-mouse or rabbit Ig (ZSGB-BIO). GAPDH was detected as a loading control.
Gata2
Manufactured by Proteintech
Sourced in United States
GATA2 is a protein that functions as a transcription factor, regulating the expression of genes involved in hematopoiesis, the process of blood cell formation. It plays a crucial role in the development and maintenance of various blood cell types, including red blood cells, white blood cells, and platelets.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (3 mentions)
α tubulin, by Proteintech (1 mentions)
Phenylmethylsulfonyl fluoride, by Wuhan Servicebio Technology (1 mentions)
Proteinase inhibitor cocktail, by Wuhan Servicebio Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Dylight 800 conjugate, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ecl reagent, by Proteintech (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
5 protocols using gata2
1
Quantifying Protein Expression in Cells
2
Overexpression and Knockdown of Key Regulatory Proteins
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From TsingKe Bio, overexpression plasmids AC092894.1, flag-USP3, HA-AR, and their associated control plasmids were bought to Tech. Co. (Beijing, China). The shRNA of AC092894.1 was purchased from Tech. Co. (Beijing, China) and Sequence in Table S3 . Additionally, siRNA for USP3 and RASGRP3 ended up being bought from Ruibo Bio. Co. (Guangzhou, China). AR siRNA was purchased from TsingKe Bio. Tech. Co. The RASGRP3 promoter plasmid ended up being bought from Bio. Tech. Co. Antibodies against p38 (ET1702-65), p-p38 (ER2001-52), and FOXP3 (ET1702-12) were purchased from HUABIO. Proteintech was used to acquire the following antibodies: GAPDH (60,004–1-lg), ATF2 (14,834–1-AP), RASGRP3 (13,162–1-AP), α-Tubulin (11,224–1-AP), GATA-2 (11,103–1-AP), RXRα (21,218–1-AP), and USP3 (12,490–1-AP). Cell Signaling Tech. Co. sold us immune responses against C-PARP (Lot No. 5625) and AR (Lot No. 19672).
3
Western Blot Analysis of Protein Expression
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RIPA lysis buffer (Sigma, St. Louis, MO, USA) containing a proteinase inhibitor cocktail (Servicebio, Wuhan, China) and phenylmethylsulfonyl fluoride (Servicebio) was used to extract total protein in tissues. Samples with the same protein concentration were resolved on a 12% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). Membranes were blocked with protein-free rapid sealing solution (Servicebio) at room temperature for 15 min, incubated with the following antibodies at 4 ℃ overnight: GATA2 (1:1,000, Proteintech Cat# 11103-1-AP, RRID:AB_10914503), TFAP2A (1:1,000, Affinity Biosciences Cat# AF0535, RRID:AB_2834124), LMBRD1 (1:1,000, ABclonal Cat# A15866, RRID:AB_2763294), KRT8 (1:2,000, Proteintech Cat# 27105-1-AP, RRID:AB_2918117). The membranes were washed with Tris-buffered saline (100 mM NaCl, 50 mM Tris-HCl, pH 7.6) containing 0.1% Tween 20 thrice for 10 min each, probed with anti-rabbit immunoglobulin G (heavy + light) [IgG (H + L)] (DyLight 800 Conjugate) (1:30,000, Cell Signaling Technology Cat# 5151, RRID:AB_10697505) for 2 h at room temperature, and washed again. The membranes were scanned and imaged using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The intensity of each band was quantitatively determined by the ImageJ analysis system (Wayne Rasband, National Institutes of Health, USA).
4
RT-qPCR and Western Blot Assay Protocol
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RT‐qPCR and WB were performed according to a previous study [28 (link)]. RT‐qPCR was performed on an iQ5 real‐time detection system using the SYBR Kit (TaKaRa, Tokyo, Japan). Relative expression levels of genes among different samples were calculated using the ΔΔCt method, with GAPDH being used as the normalization control. The primer sequences are listed in supplementary material, Table S3 . As for the WB assay, all blots were incubated with primary and secondary antibodies (TransGen Biotech, Beijing, PR China; 1:10,000) for 1 h. After incubating with ECL reagents (Proteintech, Wuhan, PR China), the blots were visualized using E‐blot. The primary antibodies recognized CARD11 (Proteintech; No. 28483‐1‐AP; 1:1,000), GATA2 (Proteintech; No. 11103‐1‐AP; 1:1,000), and β‐tubulin (Abiocode, Agoura Hills, CA, USA; No. R0742‐3; 1:1,000).
5
Comprehensive Protein Expression Analysis
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Cells were lyzed by RIPA buffer with protease inhibitor cocktail tablet (04693116001; Roche, Basel, Switzerland). 10–40 μg protein lysates were loaded to SDS-PAGE and transferred to PVDF membranes (10600021; GE Healthcare, Boston, MA). After the membranes were blocked by 5% non-fatty milk for 30 min, overnight incubation of primary antibodies, including ST6GALNAC5 (1:1000, A17782; ABclonal, Wuhan, China), GATA2 (1:1000, 11103–1-AP; Proteintech), p44/42 MAPK (ERK1/2; 1:1000, #4695; Cell Signaling Technology, Danvers, MA), phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204; 1:1000, #4370; Cell Signaling Technology), GAPDH (1:20,000, 60004–1-Ig; Proteintech) and mouse monoclonal anti-Flag (1:1000, #F1804; Sigma), was performed at 4 ℃. After incubation with corresponding secondary antibodies, the blots were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate (34578; Thermo Scientific, Waltham, MA).
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