The ChIP assay was performed in biological replicates as previously described [48 (link)]. The chromatin was sheared using a Covaris S2 instrument with 10% duty cycle, 5 intensity, 200 cycles per burst, frequency sweeping mode, 60 sec process time and for four cycles. The pull-down was performed using a RUNX1 antibody (Cell Signaling #4334). The pull-down and input control sequencing libraries were generated using the NEXTflex Rapid DNA Sequencing Kit (Bioo Scientific #5144-02) and were sequenced using SE100 reads with a HiSeq 2000 instrument. The adapters were trimmed from the sequencing reads, and the reads were aligned to the hg19 human genome using the Bowtie2 tool [49 (link)]. Quality controls, peak calling, and peak annotation was performed using the HOMER suite using the “-style factor” option, using the input samples as a background normalization control [50 (link)]. The ChIP-seq peaks that were reproducible across the biological replicates were used for downstream analysis. De novo motif analysis was performed using the MEME-ChIP suite [51 (link)].
Runx1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The RUNX1 antibody is a laboratory research tool used to detect and study the RUNX1 transcription factor. RUNX1 is a key regulator of hematopoiesis and is involved in various cellular processes. The antibody can be used in applications such as Western blotting, immunohistochemistry, and immunofluorescence to help researchers investigate the expression and localization of RUNX1 in various cell and tissue samples.
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Lab products found in correlation
Sox17 antibody, by R&D Systems (2 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Nextflex rapid dna sequencing kit, by PSC Biotech (1 mentions)
S2 instrument, by Covaris (1 mentions)
Phenol chloroform, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Formaldehyde, by Bio-Rad (1 mentions)
Chip assay kit, by Merck Group (1 mentions)
Axioimager microscope, by Hamamatsu Photonics (1 mentions)
Ccd camera, by Hamamatsu Photonics (1 mentions)
Dig nick translation mix, by Roche (1 mentions)
Large construct kit, by Qiagen (1 mentions)
P53 antibody, by Abcam (1 mentions)
Bax antibody, by Cell Signaling Technology (1 mentions)
Puma antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Lamin b antibody, by Santa Cruz Biotechnology (1 mentions)
8 protocols using runx1 antibody
1
RUNX1 ChIP-seq Protocol
2
ChIP-seq Analysis of miR-582-5p Regulation
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ChIP analysis was conducted using ChIP assay kit (Millipore, 17-610) based on the manufacturer’s instructions. A total of 1×107–5×107 cells were collected and 1% formaldehyde (Bio-Rad, CA, USA) was used to crosslink the proteins to the DNA for 25 min. Chromatin was sheared to fragments with size of 100–500 bp by sonicating the lysate. After dislodged insoluble substance by centrifugation, 100 μl DNA/protein complexes were taken as input. The samples were incubated with RUNX1 antibody (Cell Signaling Technology), normal rabbit IgG antibodies (Cell Signaling Technology), and protein A/G beads overnight at 4°C. After incubation at 65 °C for 4 h, the crosslinking of input and the samples were reversed. Then, phenol/chloroform (Invitrogen) was involved to recover DNA from the samples. Promoter binding was evaluated via PCR with primers of miR-582-5p upstream region.
3
Immunofluorescence and RNA FISH for RUNX1
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Cells were grown on Matrigel-coated coverslips for IF and RNA FISH. Detection of RUNX1 protein was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336). Staining was performed using a fluorescent secondary antibody to rabbit polyclonal antibodies and a goat anti-rabbit immunoglobulin G (IgG) (H + L) secondary antibody, Alexa Fluor 488 conjugate (Thermo Fisher Scientific #A-11001). For RNA FISH, a RUNX1 probe was created using a BAC clone (RP11-299D9) spanning the RUNX1 gene locus, obtained from the BAC/PAC Resources at the Children's Hospital Oakland Research Institute from the RPCI-11 Human Male BAC Library (Osoegawa et al., 1998 (link), Osoegawa et al., 2001 (link)). The BAC clone was amplified and isolated using the Qiagen Large-Construct Kit, and labeled by nick translation using the DIG-Nick Translation Mix (Roche #11745816910). Hybridization and detection were carried out as previously reported (Byron et al., 2013 ), with the addition of a pepsin digest (5 mg/mL pepsin in a 1:2,000 dilution in 0.01 N HCl) to allow full penetrance of probe. Hybridization occurred overnight at 37°C. A rhodamine anti-digoxygenin secondary antibody (Roche #11207750910) was used for signal detection. Images were taken on a Zeiss AxioImager microscope equipped with a Hamamatsu CCD camera and Metamorph imaging software.
4
Cardiac Tissue Protein Extraction and Western Blot
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Protein was extracted from cardiac tissues or cell samples using RIPA lysis buffer. The cell lysate was fractionated by 10% SDS‐PAGE and subsequently transferred to PVDF membranes (Millipore) and incubated with different primary antibodies overnight. After incubated with the primary antibodies, membranes were washed with TBS‐T and incubated with the corresponding secondary antibodies for 1h at room temperature. Finally, the blots were scanned by a Bio‐Rad ChemiDoc XRS+system (Bio‐Rad). The following antibodies were used: Runx1 antibody (Cell Signaling Technology; #4334; dilution 1:1000), p53 antibody (Abcam; #ab26; dilution 1:1000), p‐p53 antibody (Abcam; #ab223868; dilution 1:2000), Noxa antibody (Cell Signaling Technology; #14766; dilution 1:1000), Bax antibody (Cell Signaling Technology; #5023; dilution 1:1000), Puma antibody (Cell Signaling Technology; #24633; dilution 1:1000), Anp antibody (Abcam; #ab181242; dilution 1:10 000) and Gapdh antibody (Abcam; #ab8245; dilution 1:50 000).
5
ChIP-qPCR Analysis of Transcription Factors
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Briefly, HUAEC or E11 CD31-APC+ cells were cross-linked with 1% formaldehyde, quenched with 0.125 M glycine and resuspended in lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% triton X-100 in double distilled water) containing protease inhibitors. The chromatin solution was sonicated, and the supernatant diluted 10-fold. An aliquot of total diluted lysate was used for input genomic DNA control. Primary antibody or IgG control was incubated with Pierce Protein A/G Magnetic Beads (Thermo Scientific, 88803) at 4 °C overnight to preclear the sample. Sox17 antibody (R&D Systems, AF1924) was used to ChIP in both sorted ECs and HUAEC samples, while Runx1 antibody (Cell Signalling, D4A6) was used to perform ChIP in HUAECs. The magnetic bead coated by the antibody was washed (PBS, 0.1% Triton X-100) then incubated with the precleared sample at 4 °C overnight. The precipitates were washed, and the chromatin complexes were eluted. After reversal of cross-linking (65 °C for 4 h), the DNA was purified using QIAquick PCR purification kit (Qiagen, 28104) and 100 pg was used as a template in each quantitative PCR reaction for quantitative analysis. Oligonucleotides used in PCR for quantitative ChIP are listed in Supplementary Table 3 . Antibody dilutions are listed in Supplementary Table 7 .
6
Jurkat Cell RUNX1 Knockdown Protocol
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Jurkat cells were electroporated with 80nM of siRNA against human RUNX1 using the Amaxa Nucleofector system (Lonza Inc., Allendale, NJ, USA). Multiple siRNA targeting RUNX1 mRNA (Dharmacon Inc, Lafayette, CO) were initially screened. Only the siRNA with greater than 50% knock down efficiency of RUNX1 protein, were used for subsequent experiment. Briefly, 1×106 Jurkat cells were electroporated with either a control or two different RUNX1 siRNA (siR1, siR2) for 18 hours. Cells were harvested for either ChIP or western blot analysis.
Blots were probed with polyclonal RUNX1 antibody (Cell Signaling Technology Inc, Beverly, MA) or SCL antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). Blots were stripped and re-probed with polyclonal Actin, Lamin B antibody or mouse monoclonal GAPDH antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). Protein were detected using species matched HPR-conjugated secondary antibodies and chemiluminiscence imagining.
Blots were probed with polyclonal RUNX1 antibody (Cell Signaling Technology Inc, Beverly, MA) or SCL antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). Blots were stripped and re-probed with polyclonal Actin, Lamin B antibody or mouse monoclonal GAPDH antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). Protein were detected using species matched HPR-conjugated secondary antibodies and chemiluminiscence imagining.
7
Chromatin Immunoprecipitation of EC Transcription Factors
8
Immunofluorescence Staining of Cell Markers
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Cells grown on coverslips were fixed with using 3.7% formaldehyde in phosphate buffered saline (PBS) for 10 min. Cells were then permeabilized in 0.1% Triton X-100 in PBS, and washed in 0.5% Bovine Serum Albumin in PBS. Detection was performed using a rabbit polyclonal Runx1 antibody (Cell Signaling Technology #4336), a mouse monoclonal Vimentin (Santa Cruz Biotechnology sc-6260), a rabbit polyclonal N-cadherin (Santa Cruz Biotechnology sc-7939) and a mouse monoclonal to E-cadherin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Staining was performed using fluorescent secondary antibodies; for rabbit polyclonal antibodies a goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate (Life Technologies A-11008), was used and for mouse monoclonal a F(ab')2-goat anti-mouse IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate was used (Life Technologies A-11001).
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