Revision 8

#53528Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

50

Source/Isotype:

Rabbit IgG

UniProt ID:

#P55317

Entrez-Gene Id:

3169

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunofluorescence (Immunocytochemistry) 1:1600 - 1:3200
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

FoxA1/HNF3α (E7E8W) Rabbit mAb recognizes endogenous levels of total FoxA1/HNF3α protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human FoxA1/HNF3α protein.

Background

Forkhead box protein A1 (FoxA1, HNF3α) is a transcription factor required for the development of endoderm-derived organs, such as liver, lung, and prostate (1). FoxA1 functions as a pioneer factor that is recruited primarily to the distant enhancers to change chromatin structure for transcription in a cell type-specific manner (2,3). The FoxA1 transcription factor is implicated in various diseases, playing a role in hormone-dependent disorders, such as breast and prostate cancers (4). The treatment of relapsing-remitting multiple sclerosis patients with IFN-β results in FoxA1-induced stimulation of a novel population of FoxA1(+) regulatory T cells, suggesting a possible immunosuppressive role for FoxA1 (5).

  1. Friedman, J.R. and Kaestner, K.H. (2006) Cell Mol Life Sci 63, 2317-28.
  2. Lupien, M. et al. (2008) Cell 132, 958-70.
  3. Wang, A. et al. (2015) Cell Stem Cell 16, 386-99.
  4. Augello, M.A. et al. (2011) EMBO J 30, 3885-94.
  5. Liu, Y. et al. (2014) Nat Med 20, 272-82.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 8
#53528

FoxA1/HNF3α (E7E8W) Rabbit mAb

Western Blotting Image 1: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using FoxA1/HNF3α (E7E8W) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
Immunoprecipitation of FoxA1/HNF3α from Hep G2 extract. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is FoxA1/HNF3α (E7E8W) Rabbit mAb. Western blot analysis was performed usingFoxA1/HNF3α (E7E8W) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody.
Immunofluorescence Image 1: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of MCF7 cells (left, positive) and SK-MEL-5 cells (right, negative) using FOXA1 (E7E8W) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
Flow Cytometry Image 1: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
Flow cytometric analysis of fixed/permeabilized Daudi cells (blue, negative) and MCF7 cells (green, positive) using FoxA1/HNF3α (E7E8W) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from LNCaP cells and FoxA1/HNF3α (E7E8W) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across KLK3.
Chromatin Immunoprecipitation Image 2: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin LNCaP cells and FoxA1/HNF3α (E7E8W) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 19 (upper), including KLK3 (lower), a known target of FoxA1/HNF3α.
Chromatin Immunoprecipitation Image 3: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from untreated LNCaP cells and either FoxA1/HNF3α (E7E8W) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human SLUG exon 3 primers, human PSA enhancer primers, SimpleChIP® Human KLK3 Promoter Primers #32784, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT and RUN Image 1: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
CUT&RUN was performed with LNCaP cells and FoxA1/HNF3α (E7E8W) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across RABL3, a known target gene of FoxA1 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
CUT&RUN was performed with LNCaP cells and FoxA1/HNF3α (E7E8W) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 3 (upper), including RABL3 (lower), a known target gene of FoxA1 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: FoxA1/HNF3α (E7E8W) Rabbit mAb Expand Image
CUT&RUN was performed with LNCaP cells and either FoxA1/HNF3α (E7E8W) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human RABL3 promoter primers, human CCDC190 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.