Before the collection of protein extracts, cells were dissolved in lysis buffer. The cell lysis buffer was mixed with Flag M2-affinity gel-conjugated GATA1 antibody at 4°C for 12 h. After that, the protein was gradually eluted by KCl-contained buffer. The lysates (approximately 300 µg protein) were incubated with fresh protein A-beads (35 µL, #9863, CST) and 1 µg anti-GATA1 (1:1,000, ab181544, Abcam), anti-Flag (1:500, AF519, Beyotime) or IgG (1:500, ab172730, Abcam) at 4°C for 3 h. The magnetic beads were lysed in lysis buffer and loaded in SDS-PAGE. The immunoprecipitated proteins were reacted with anti-HDAC1 (1:1,000, ab109411, Abcam), anti-HDAC2 (1:1,200, ab32117, Abcam) and anti-HDAC3 (1:2,000, ab32369, Abcam). The immunoblot reactions were performed as described previously [25 (link)].
Ab181544
Manufactured by Abcam
Sourced in United States
Ab181544 is an antibody product offered by Abcam. It is a primary antibody that can be used for research purposes. The core function of this product is to bind to a specific target, but further details on its intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab92360, by Abcam (2 mentions)
Ab137096, by Abcam (2 mentions)
Hc201 01, by Transgene (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ab109411, by Abcam (1 mentions)
Ab32117, by Abcam (1 mentions)
Ab32369, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Af519, by Beyotime (1 mentions)
Antibodies against cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ecl western blotting system, by GE Healthcare (1 mentions)
Promoter binding tf profiling plate array 1, by Signosis (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (1 mentions)
Chip assay kit, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
D glucose, by Merck Group (1 mentions)
Runx3, by Thermo Fisher Scientific (1 mentions)
Magnetic protein a and g beads, by Thermo Fisher Scientific (1 mentions)
Ab137332, by Abcam (1 mentions)
7 protocols using ab181544
1
Immunoprecipitation and Immunoblotting for GATA1
2
Western Blot Protein Analysis
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Cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate, and 1 mM dithiothreitol) supplemented with protease inhibitors (Roche, Basel, Switzerland) and incubated for 30 min on ice. After centrifugation at 18,800 g for 10 min, the protein concentration in the supernatant was determined using the BCA method. Equal amounts of protein were separated by sodium dodecyl sulfate (SDS)-sulfate-polyacrylamide gel electrophoresis in Tris/glycine/SDS running buffer. The separated proteins were transferred onto a polyvinylidene fluoride membrane in Tris-glycine transfer buffer that was blocked with 3% Tris-buffered saline with Tween 20 (BSA-TBST) for 1 h at room temperature. The membranes were probed with primary RanGAP1 antibody (ab92360, Abcam) at a 1:500 dilution and HBG2 (ab137096, Abcam), GATA1 (ab181544, Abcam), and β-actin antibodies (HC201-01, TransGen Biotech, Beijing, China) at a 1:1,000 dilution in 3% BSA-TBST at 4°C overnight. The membranes were subsequently rinsed with TBST three times for 10 min each and incubated with a secondary antibody for 1 h at room temperature. Finally, the membranes were rinsed three times with TBST solution and imaged using an Image Quant ECL machine (Tanon 5200, Tanon, Shanghai, China).
3
Endothelial Cell Culture and Apoptosis Analysis
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Human Endothelial Culture Medium was purchased from Thermo Scientific (Grand Island, NY, USA). The FITC-Annexin V/PI Apoptosis Detection Kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). The Promoter-Binding TF Profiling Plate Array I (FA-2001) was obtained from Signosis (Santa Clara, CA, USA). Dual-Luciferase Reporter Assay System (E1960), PGL4.12 (E6671) and PGL4.73 (E6911) vectors were purchased from Promega (Madison, WI, USA). The ChIP assay kit was obtained from Millipore (Billerica, MA, USA). Plasmid pcDNA3.1 (+), a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) kit and TRIzol Reagent were purchased from Invitrogen (Grand Island, NY, USA). Antibodies against CREG (ab191909, ab68341) and GATA1 (ab181544) were purchased from Abcam (Cambridge, MA, USA). Antibodies against cleaved caspase-3 and GAPDH were obtained from Cell Signalling Technology (Danvers, MA, USA). The enhanced chemiluminescence (ECL) Western Blotting System was purchased from GE Life Sciences (Marlborough, MA, USA). D-glucose, free fatty acid (FFA)-free bovine serum albumin (BSA), palmitate and other chemicals, if not specified otherwise, were purchased from Sigma-Aldrich (St Louis, MO, USA).
4
ChIP-seq Protocol for Runx3 and GATA1
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108 cells were crosslinked with 2 mM DSG for 30 min and 1% FA for 30 min. Cells were lysed (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1(+ protease inhibitors) and sonicated on the Covaris to give fragments of 100–300 bp. Samples were diluted (0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1,167 mM NaCl (+ protease inhibitors) and incubated over night with 1 µL of Runx3 (39301 Active motif/ ThermoFisher) and 2 µL of Runx3 (653604 BioLegend) or 2 µL Recombinant Anti-GATA1 antibody (EPR17362) - ChIP Grade (ab181544, Abcam). Magnetic protein A and G beads (26162, ThermoFisher Scientific) were used to isolate antibody-chromatin complexes. Samples were washed three times with with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and then with TE + 50 mM NaCl before elution (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) following by RNase and proteinase K treatment and DNA purification. For information regarding the replicates see Supplementary Table 4 .
5
Immunoblotting Reagents and Antibodies
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Antibodies against Fli-1 (ab153909, 1:1000), Rb (ab181616, 1:2000), MDM2 (ab38618, 1:1000), Gata-1 (ab181544, 1:1000), Bcl-2 (ab32124, 1:1000), p110 (ab151549, 1:1000), SHIP-1 (ab45142, 1:1000), MMP-1 (ab137332, 1:1000), TGF-β2 (ab113670, 1:1000), phospho-ERK 1/2 (T202 +Y204) (ab223500, 1:1000), and ERK1 (ab32537, 1:1000) were purchased from Abcam (Cambridge, UK). Antibodies against MMP-9 (#13667s, 1:1000), ICAM-1 (#4915s, 1:1000), GAPDH (#2118s, 1:1000), and VEGF-1 (#2893s, 1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primers (Rb, MDM2, Gata-1, Bcl-2, PI3 K/Ras, SHIP-1, MMP-1, TGF-β2, ERK1, MMP-9, ICAM-1, and VEGF-1) were purchased from GenScript (Nanjing, China). Anti-rabbit and anti-mouse LgG (H+L) [Dylight (TM) 800 4 × PEG Conjugate] secondary antibodies used in this study were purchased from Cell Signaling Technology and used at a 1:30000 dilution in experiments.
6
Quantifying Cellular Protein Expression Using SDS-PAGE and Western Blotting
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Total protein from cells was isolated using the radio-immunoprecipitation assay lysis buffer (P0013C, Beyotime Biotechnology Co., Ltd., Shanghai, China) containing phenylmethylsulfonyl fluoride. After concentration examination by a bicinchoninic acid kit (Thermo Fisher Scientific), an equal amount of protein sample (50 μg) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and loaded onto polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). After that, the membranes were blocked by 5% nonfat milk for 1 h and reacted with the antibodies against NRBP2 (1:1,000, PA5-65039, Thermo Fisher Scientific), GATA1 (1:1,000, ab181544, Abcam Inc., Cambridge, MA, USA) and GAPDH (1:2,500, ab181602, Abcam) at 4°C overnight, and then with goat anti-rabbit immunoglobulin G (IgG) (1:2,000, ab97051, Abcam) at 22–25°C for 1 h. The protein blots were visualized using an enhanced chemiluminescence kit (P0018FS; Beyotime Biotechnology Co., Ltd., Shanghai, China) and photographed by an image analyzing system (Bio-Rad Laboratories, CA, USA). The intensity of the protein signals was determined using the Quantum One v.4.6.2 software with GAPDH as the internal loading [20 (link)].
7
Western Blot Analysis of Cellular Proteins
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Cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate, 1 mM dithiothreitol) supplemented with protease inhibitors (Roche, Basel, Switzerland) and incubated for 30 min on ice. After centrifugation at 18,800 ×g for 10 min, the protein concentration in the supernatant was determined. Equal amounts of protein were separated using sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis in a Tris/glycine/SDS running buffer. The separated proteins were transferred onto a polyvinylidene fluoride membrane in Tris/glycine transfer buffer, which was then blocked with 3% BSA-TBST (Tris buffered saline with Tween 20) for 1 h at room temperature. The membranes were probed with primary RanGAP1 mouse monoclonal antibody (ab92360, Abcam) at a 1:500 dilution and HBG2 (ab137096, Abcam), GATA1 (ab181544, Abcam), and β-actin mouse monoclonal antibodies (HC201-01, TransGen Biotech, Beijing, China) at a 1:1000 dilution in 3% BSA-TBST at 4℃ overnight. The membranes were subsequently rinsed with TBST thrice for 10 min each and incubated with secondary antibody for 1 h at room temperature. Finally, the membranes were rinsed with TBST solution thrice and imaged using an Image Quant ECL machine (Tanon 5200, Tanon, Shanghai, China).
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