ChIP assay was performed using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer’s instructions. Briefly, CD4+ T cells were cross-linked with 1% formaldehyde for 10 min at room temperature, harvested, and resuspended in lysis buffer. Following sonication and centrifugation, sheared chromatin was incubated overnight at 4 °C with anti-Foxp1 (Abcam, USA). Serum IgG served as a negative control.
Anti foxp1
Manufactured by Abcam
Sourced in United States
Anti-FOXP1 is a laboratory reagent used for the detection and quantification of the FOXP1 protein in biological samples. FOXP1 is a transcription factor that plays a role in the regulation of gene expression. This product can be used in various research applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of FOXP1 in cells and tissues.
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Lab products found in correlation
Anti e cadherin, by Abcam (2 mentions)
Anti β gal, by Promega (2 mentions)
Anti gfap, by Agilent Technologies (2 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Anti erα, by Abcam (1 mentions)
Anti kras, by Proteintech (1 mentions)
Anti pten, by Proteintech (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti foxp2, by Abcam (1 mentions)
Anti ctbp1, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg, by Abcam (1 mentions)
Ecl detection kit, by Beyotime (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Anti gadph, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
10 protocols using anti foxp1
1
ChIP Assay for Foxp1 Binding
2
Western Blot Analysis of Protein Targets
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Protein samples (10 μg) were separated on a 12.5% sodium dodecyl sulfate polyacrylamide gel and loaded and run at 30 mA for 120 min. Samples were then blotted onto 0.45-μm nitrocellulose membranes (Catalog number 162–0114, Bio-Rad, Hercules, CA). The membranes were incubated overnight at 4 °C with the following diluted antibodies: anti-KRAS (Catalog number 60309-1-Ig, Proteintech, Chicago, IL) at 1:500, anti-PTEN (Catalog number 22034-1-AP, Proteintech) at 1:250, anti-FOXP1 (Catalog number ab16645, Abcam) at 1:500, anti-ERα (Catalog number; ab17087, Abcam) at 1:100 and anti-β-actin (Catalog number A5441, Sigma Aldrich) at 1:500. The membranes were then incubated with anti-mouse or rabbit IgG HRP-linked Antibody (Catalog number 7076, Cell Signaling Technology, Beverly, MA). Protein bands on the same membranes were visualized using enhanced chemiluminescence (ChemiDoc XRS, Bio-Rad) according to the manufacturer’s protocol. Band intensity was analyzed with Molecular Imager, Image Lab Ver.3.0.1 (Bio-Rad).
3
IHC Analysis of HCC Tissue Markers
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Sectioned HCC tissues from patients or mice were incubated with 3% hydrogen peroxide and immersed with 0.05% Tween 20 in Tris-EDTA buffer. After incubation in 4% dry milk and 0.3% goat serum, the sections were incubated overnight with anti-FOXP1, anti-Ki67, anti-E-cadherin, or anti-N-cadherin primary antibodies (Abcam). Slides were imaged under a light microscope (Olympus) after incubation with HRP goat antirabbit IgG secondary antibody.
4
Chromatin Immunoprecipitation of Histone Modifications
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The ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (CST) according to the manufacturer’s protocol. Immunoprecipitants were recovered overnight with specific antibodies or with normal rabbit IgG (CST). The purified DNA was amplified by PCR using the SP-B promoter-specific primers: Sftpb forward, 5′-CAGACAGAAGTCATCCTTGTTGAATG-3′; Sftpb reverse, 5′-ACATGGTACCGACTTGGCC-3′. PCR was performed under the following conditions: 95 °C for 5 min, followed by 30–35 cycles of 95 °C for 30 sec, 58 °C for 30 sec and 72 °C for 30 sec, and a final step of 72 °C for 5 min. The PCR products were subjected to 2% agarose gel electrophoresis. The following rabbit antibodies were used: anti-Histone H3 (CST; 1 μg/sample), anti-Pan-Methyl-Histone H3 (K9) (CST; 1 μg/sample), anti-Acetyl-Histone H3 (K9) (CST; 1 μg/sample), anti-CtBP1 (Abcam; 1 μg/sample), anti-Foxp1 (CST; 1 μg/sample), and anti-Foxp2 (Abcam; 1 μg/sample).
5
Western Blot Analysis of FOXP1 Protein
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The total protein was extracted using the RIPA buffer (Sigma–Aldrich, St. Louis, MO) supplemented with protease inhibitors cocktail (Roche, Diagnostics, Mannheim, Germany). Protein concentration was measured using BCA assay. Proteins were separated by SDS–PAGE, followed by being transferred to PVDF membrane (Millipore, Bedford, MA). After blocked with 5% non-fat milk, the membrane was incubated with the primary antibodies, including anti-FOXP1 (1:1000, Abcam, Cambridge, MA), anti-GADPH (1:1000, Abcam) d. After washing with TBST, PVDF membrane was incubated with HRP-conjugated goat anti-rabbit IgG (Abcam) at room temperature for 2 h. Finally, the films were developed using ECL detection kit (Beyotime Biotechnology, Shanghai, China).
6
Western Blot Analysis of FOXP1 in N2a Cells
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Proteins from lysed N2a cells were denatured and loaded on sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the separated proteins were transferred to PVDF membranes, blocked in TBS-T (150 mM NaCl, 10 mM TRIS-HCl pH 7.5, and 0.1% Tween 20) containing 5% (w/v) dry milk, and stained with anti-FOXP1 (1:1000, Abcam) and anti-GAPDH (1:500, Santa Cruz) antibodies. The specific bands were analyzed by immunoblot infrared imaging system (LI-COR Biosciences) after incubation with the corresponding secondary antibodies. The density of each band was measured using NIH ImageJ and subtracted from the nearby background.
7
Molecular Markers for Neuronal Lineages
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Plasmid pCAG-dsRed was from Addgene (Plasmid #11151). Neurod1-Cre was provided by Dr. Franck Polleux (Columbia University). Pak3K297R was gifted by Dr. Rick Horwitz (University of Virginia). The PAK3 mutant was subcloned into the pCMVmyc vector.
The following primary antibodies were used: anti-EZH2 1:500 (BD Biosciences, San Jose, USA, Cat. No. 612666), anti-EZH2 1:1000 (Cell Signaling, Danvers, USA, Cat. No. #5246), anti-GAPDH 1:5000 (Sigma-Aldrich, St. Louis, USA, Cat. No. G8795), anti-GFP 1:1200 (Thermo Fisher Scientific, Waltham, USA, Cat. No. A10262), anti-TUJ1 1:1000 (Biolegend, San Diego, USA, Cat. No. 801201), anti-H3K27me3 1:2000 (Millipore, Burlington, USA, Cat. No. 07449), anti-H3 1:1000 (Cell Signaling, Cat. No. #4499), anti-dsRED 1:1000 (Clontech, Mountain View, USA, Cat. No. 632496), anti-Tbr1 1:1000 (Abcam, Waltham, USA, Cat. No. ab31940), anti-Ctip2 1:500 (Abcam, Cat. No. ab18465), anti-Foxp1 1:1000 (Abcam, Cat. No. ab16645), and anti-Cre 1:1000 (Millipore, Cat. No. MAB3120).
The following primary antibodies were used: anti-EZH2 1:500 (BD Biosciences, San Jose, USA, Cat. No. 612666), anti-EZH2 1:1000 (Cell Signaling, Danvers, USA, Cat. No. #5246), anti-GAPDH 1:5000 (Sigma-Aldrich, St. Louis, USA, Cat. No. G8795), anti-GFP 1:1200 (Thermo Fisher Scientific, Waltham, USA, Cat. No. A10262), anti-TUJ1 1:1000 (Biolegend, San Diego, USA, Cat. No. 801201), anti-H3K27me3 1:2000 (Millipore, Burlington, USA, Cat. No. 07449), anti-H3 1:1000 (Cell Signaling, Cat. No. #4499), anti-dsRED 1:1000 (Clontech, Mountain View, USA, Cat. No. 632496), anti-Tbr1 1:1000 (Abcam, Waltham, USA, Cat. No. ab31940), anti-Ctip2 1:500 (Abcam, Cat. No. ab18465), anti-Foxp1 1:1000 (Abcam, Cat. No. ab16645), and anti-Cre 1:1000 (Millipore, Cat. No. MAB3120).
8
Western Blot Analysis of Protein Expression
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Proteins extracted from SK-HEP-1 or Huh7 cells (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membrane. Membrane was incubated overnight with anti-FOXP1, anti-proliferating cell nuclear antigen (PCNA), anti-c-Myc, anti-Bcl-2, anti-cleaved caspase-3, anti-E-cadherin, anti-N-cadherin, or anti-β-actin antibodies (Abcam) at 4 °C and then incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:5000; Abcam). Immunoreactivities were determined by enhanced chemiluminescence (KeyGen, Nanjing, China).
9
Immunocytochemical Characterization of Differentiated Cells
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Cells were plated and fixed after 4 hours (as precursors) or 7 d (as differentiated cells), in 4% formaldehyde or 0.2% glutaraldehyde in 4% formaldehyde. Fluorescent immunocytochemistry was performed using standard protocols using the following primary antibodies: anti-β-Gal (1:1000, Promega), anti-Nestin (1:400, BD Pharm), anti-Oct4 (1:100, Santa Cruz), anti-Pax6 (1:50, DSHB), anti-Nkx2.1 (1:100, DAKO), anti-Mash1 (1:200, BDPharm), anti-β-III-tubulin (mouse 1:1000; rabbit 1:500, both Sigma), anti-GFAP (1:2000, DAKO), anti-GABA (1:500, Sigma), anti-DARPP32 (1:20,000, a gift from Paul Greengard) and anti-FoxP1 (1:500, Abcam). Secondary antibodies used were Alexa-fluor α 594 anti-mouse and α 488 anti-rabbit (both 1:200, DAKO).
Cells were visualised under UV fluorescence using a Leitz microscope and cell counts were performed using a grid randomly placed over 5 different fields per coverslip. The number of positive cells counted was expressed as mean ± SEM from replicates of 3–4 coverslips per condition. Images were processed using Optronics MagnaFire Software and Adobe Photoshop.
Cells were visualised under UV fluorescence using a Leitz microscope and cell counts were performed using a grid randomly placed over 5 different fields per coverslip. The number of positive cells counted was expressed as mean ± SEM from replicates of 3–4 coverslips per condition. Images were processed using Optronics MagnaFire Software and Adobe Photoshop.
10
Immunohistochemical Staining of Rat Brain
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Rats were transcardially perfused and tissue was prepared, as previously described.44 (link) Sections were processed for Nissl staining using cresyl violet and immunohistochemistry with the following antibodies: anti-β-Gal (1:1000, Promega), anti-GFAP (1:2000, DAKO), anti-NeuN (1:4000, Chemicon), anti-doublecortin (DCX, 1:500, Abcam), anti-DARPP32 (1:10,000, a gift from Paul Greengard), anti-FoxP1 (1:500, Abcam), anti-calbindin (1:20,000, Sigma) and anti-parvalbumin (1:4000, Sigma). The basic protocol was the same for each antibody.
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