The following antibodies were used: anti-FLAG (Sigma, F7425), anti-IKZF3 (Cell Signaling, #15103), anti-IKZF1 (Cell Signaling, #9034), anti-RUNX1 (Abcam, ab92336), anti-RUNX3 (Cell Signaling, #9647), anti-CBFβ (Santa Cruz, sc-20693), anti-CRBN (Sigma, HPA045910), anti-ubiquitin (K48) (EMD Millipore, #05–1307), anti-TUBULIN (Santa Cruz, sc-8035), anti-VINCULIN (Santa Cruz, sc-73614), anti-Rabbit IgG-HRP (GE Healthcare, NA934V), and anti-mouse IgG-HRP (GE Healthcare, NA931V). The following agarose beads were used: anti-FLAG M2 Affinity Gel (Sigma, A2220) and Glutathione Sepharose 4B (GE Healthcare, #17075601). The following in vitro translation kit was used: TNT T7 Coupled Reticulocyte Lysate Systems (Promega, L4610). Benzonase was used according to manufacturer (Sigma, E1014). The following compounds were used: Lenalidomide (Sigma, CDS022536), Pomalidomide (Sigma, P0018), Bortezomib (Millennium Pharmaceuticals), and AI-10–104 was kindly provided by Dr. John H. Bushweller.
Anti runx1
Manufactured by Abcam
Sourced in United States
Anti-RUNX1 is a primary antibody that specifically targets the RUNX1 protein. RUNX1 is a transcription factor that plays a crucial role in the development and function of various cell types, including hematopoietic cells. This antibody can be used for applications such as Western blotting, immunohistochemistry, and immunocytochemistry to detect and study the RUNX1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Lenalidomide, by Merck Group (2 mentions)
Anti ikzf3, by Cell Signaling Technology (2 mentions)
Anti ikzf1, by Cell Signaling Technology (2 mentions)
Anti cbfβ, by Santa Cruz Biotechnology (2 mentions)
Anti crbn, by Merck Group (2 mentions)
Anti ubiquitin k48, by Merck Group (2 mentions)
Anti vinculin, by Santa Cruz Biotechnology (2 mentions)
Anti rabbit igg hrp, by GE Healthcare (2 mentions)
Anti mouse igg hrp, by GE Healthcare (2 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (2 mentions)
Pomalidomide, by Merck Group (2 mentions)
Anti tubulin, by Santa Cruz Biotechnology (2 mentions)
Anti runx3, by Cell Signaling Technology (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Benzonase, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
12 protocols using anti runx1
1
Characterizing CRBN-mediated Protein Degradation
2
Western Blot Protein Analysis
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Cells were pelleted, washed with cold PBS, and lysed in RIPA buffer (Thermo) with protease inhibitor cocktail (Roche). 35 μg total protein was separated by SDS-PAGE and transferred to an Immobilon PVDF membrane (Pall). The membrane was blocked and incubated overnight with primary antibodies. After a final incubation with secondary antibodies conjugated with horseradish peroxidase (1:5000 dilution; Millipore), immune complexes were detected with HRP chemiluminescent substrate (Millipore). Antibodies and dilutions used were: anti-RUNX1 (1:1000, Abcam) and anti-β-actin (1:5000, Novus).
3
Protein Expression Analysis Protocol
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Cells were harvested and lysed by RAPI buffer (Beyotime, Shanghai, China), and then were incubated on ice for 30 min. The BCA kit (Beyotime, Shanghai, China) was used to quantify the concentration of protein. Then, the protein was separated with 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with the primary antibodies (anti-RUNX1, anti-MMP2 and anti-MMP9, 1: 1000, Abcam, Cambridge, MA, USA; GAPDH, 1: 1000, Santa Cruz Biotechnology, Inc., USA) overnight at 4°C. The second antibody (purchased from Beyotime, Shanghai, China) used in this study was incubated at 37 for 1 h. Finally, membranes were incubated with BeyoECL Plus (Beyotime, Shanghai, China), and detected using the ChemiDoc™ XRS+ imaging system (Bio-Rad, California, USA).
4
Protein Extraction and Western Blotting Protocol
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Total proteins of the cells were extracted by Golden lysis buffer (Tris-HCl [pH 8.0], 400 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na pyrophosphate, 1% Triton X-100, 10% glycerol), with a supplement of protease inhibitors (Roche, Indianapolis, IN, USA). The concentration of total protein was measured with a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA). 20 mg of protein was loaded onto 10% SDS-PAGE gel and was transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were then incubated with primary and secondary antibodies. Protein signals were detected via the enhanced chemiluminescence (ECL) method. The primary antibodies used in this study include anti-VEGFA (1:1,000, Abcam), anti-ZNF24 (1:1,000, Abcam), anti-GAPDH (1:2,000, Abcam), anti-Runx1 (1:1,000, Abcam), and anti-H3K27me3 (1:1,000, Abcam). All primary antibodies were incubated overnight at 4°C. The horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Sigma-Aldrich) was incubated for 2 h at room temperature.
5
Chromatin Immunoprecipitation Assay Protocol
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The chromatin immunoprecipitation assay was performed using a Pierce Magnetic ChIP Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The antibodies used for immunoprecipitation were anti-H3K27ac (CST), anti-E2F7 (Abcam), and anti-RUNX1 (Abcam). Purified DNA was detected with specific primers for the promoter region using quantitative PCR. The primer sequences used for quantitative PCR are shown in Table S6 .
6
RUNX1 Chromatin Immunoprecipitation in PAK4-overexpressing Cells
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Transfected cells were washed and cross-linked with 1% formaldehyde. The cells were sonicated to shear DNA and immunoprecipitated with anti-RUNX1 (Abcam, USA) antibodies and control immunoglobulin G at 4 °C overnight. After elution and reverse cross-linking, the purified DNA was resuspended in TE buffer. DNA samples (2 ul) were then amplified by PCR. This experiment was performed in MCF-7cells stably overexpressed PAK4 and the enrichment of target gene promoter pulled down by RUNX1 antibody.
7
Embryonic Hematopoietic Lineage Tracing
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Embryos were fixed with 2% paraformaldehyde at 4°C, equilibrated overnight at 4°C in 30% sucrose and then snap‐frozen in Tissue Tek (Sakura Finetek). Ten micrometre cryosections were prepared and stained with anti‐GFP (chicken; Life Technologies), anti‐Runx1 (rabbit; Abcam), anti‐CD34 (FITC), anti‐chicken‐Alexa647 (Millipore), anti‐rabbit‐Alexa555, anti‐Kit (goat; R&D Systems), and anti‐goat‐Alexa488 before mounting in DAPI‐containing Vectashield (Vectorlabs). UG26‐1B6 cells grown on microscope slides were stained with anti‐Gata3 (rat; Absea), anti‐rat‐biotin (BD Biosciences), and Streptavidin‐Cy5 (Jackson Immunoresearch) before mounting in DAPI‐containing Vectashield. X‐gal staining of Gata3+/lz and Runx1+/lz embryos was carried out as described previously.22 Cryosections were prepared and counterstained with Neutral Red. Brightfield images were acquired with a Zeiss AxioSkop2 Wide‐Field Microscope and fluorescent images with a Widefield Zeiss Observer and analysed using Zen software.
8
Western Blot Analysis of RUNX1, p38, and VEGF
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For Western blot analysis, HRMECs and tissue samples were lysed in RIPA buffer containing protease inhibitor cocktail (Generay, Shanghai, China). The proteins were transferred to PVDF membranes and probed with primary antibodies, including anti-RUNX1 (#ab35962; dilution, 1:1000), anti-p38 (#ab170099; dilution, 1:1000), anti-VEGF (#ab222510; dilution, 1:1000) and anti-GAPDH (#ab181602; dilution, 1:1000) or anti-H3 (#ab1791; dilution, 1:1000); all primary antibodies were purchased from Abcam (Cambridge, MA, U.S.A.). The second antibody HRP-conjugated goat anti-mouse (#ab19195; dilution, 1:10,000) and goat anti-rabbit IgG (#ab6721; dilution, 1:10,000) were used to detect the expression of T-RUNX1, T-p38, T-VEGF and T-GAPDH, respectively; all secondary antibodies were purchase from Abcam (Cambridge, MA, U.S.A.). The blots were detected using Bio-Imaging System and Quality One 1-D analysis software (Bio-Rad, Richmond, CA, U.S.A.).
9
Immunoprecipitation Assay for E3 Ligase Complexes
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The following antibodies were used: anti-FLAG (Sigma, F7425), anti-IKZF3 (Cell Signaling, #15103), anti-IKZF1 (Cell Signaling, #9034), anti-RUNX1 (Abcam, ab92336), anti-RUNX3 (Cell Signaling, #9647), anti-CBFβ (Santa Cruz, sc-20693), anti-CRBN (Sigma, HPA045910), anti-ubiquitin (K48) (EMD Millipore, #05-1307), anti-TUBULIN (Santa Cruz, sc-8035), anti-VINCULIN (Santa Cruz, sc-73614), anti-Rabbit IgG-HRP (GE Healthcare, NA934V), and anti-mouse IgG-HRP (GE Healthcare, NA931V). The following agarose beads were used: anti-FLAG M2 Affinity Gel (Sigma, A2220) and Glutathione Sepharose 4B (GE Healthcare, #17075601). The following in vitro translation kit was used: TNT T7 Coupled Reticulocyte Lysate Systems (Promega, L4610). Benzonase was used according to manufacturer (Sigma, E1014). The following compounds were used: Lenalidomide (Sigma, CDS022536), Pomalidomide (Sigma, P0018), Bortezomib (Millennium Pharmaceuticals), and AI-10-104 was kindly provided by Dr. John H. Bushweller.
10
Multiparametric Analysis of Hematopoietic Cells
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For ChIP, immunoprecipitation, and WB, anti-RUNX1 (Abcam), RAG1 (D36D3; Cell Signaling Technology), anti-FLAG (Sigma-Aldrich), anti–CBF-β (Abcam), and normal rabbit IgG (Santa Cruz Biotechnology, Inc.) were used. For immunofluorescence, RAG1 (D36D3) and RUNX1 (Abcam) were used. For flow cytometry, CD1a-FITC (NA1/34), CD13-FITC (WM47; Dako); CD3–Alexa Fluor 700 (UCHT1), CD4-V450 (RPA-T4), CD5-PerCP-Cy5.5 (L17F12), CD7-PE (M-T701), CD8-APC (RPA-T8), CD34-PerCP-Cy5.5 (8G12), CD45-V500 (H130), HLA-DR-FITC (L243), CD34-APC (8G12; BD); CD117-PECy7 (104D2D1; Beckman Coulter); and CD123-APC (AC145; Miltenyi Biotec) were used.
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