Revision 4

#5628Store 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, W-S, IP, ChIP, ChIP-seq

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

50

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q02556

Entrez-Gene Id:

3394

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 20 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.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:50
Chromatin IP 1:25
Chromatin IP-seq 1:25

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

IRF-8 (D20D8) Rabbit mAb detect endogenous levels of total IRF-8 protein. An unknown background band is detected at 80 kDa in some cell lines.

Species Reactivity:

Human, Mouse

Species predicted to react based on 100% sequence homology

Rat, Monkey, Xenopus, Bovine

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly65 of human IRF-8 protein.

Background

Interferon regulatory factors (IRFs) comprise a family of transcription factors that function within the Jak/Stat pathway to regulate interferon (IFN) and IFN-inducible gene expression in response to viral infection (1). IRFs play an important role in pathogen defense, autoimmunity, lymphocyte development, cell growth, and susceptibility to transformation. The IRF family includes nine members: IRF-1, IRF-2, IRF-9/ISGF3γ, IRF-3, IRF-4 (Pip/LSIRF/ICSAT), IRF-5, IRF-6, IRF-7, and IRF-8/ICSBP. All IRF proteins share homology in their amino-terminal DNA-binding domains. IRF family members regulate transcription through interactions with proteins that share similar DNA-binding motifs, such as IFN-stimulated response elements (ISRE), IFN consensus sequences (ICS), and IFN regulatory elements (IRF-E) (2).

IRF-8/ICSBP is expressed predominately in hematopoietic cells and is further increased upon treatment with interferon (3,4). IRF-8 can function as a transcription repressor of ICS-containing promoters (4). Expression of IRF-8 can lead to the downregulation of the anti-apoptotic protein Bcl-2 (5). Originally described as being induced by IFN-γ, IRF-8 expression is also elevated by IRF-α as well as IL-12 in NK and T cells (6). IRF-8 deficient mice have enhanced susceptibility to various pathogens and impaired production of interferons, as well as deregulated hematopoiesis that resembles chronic myelogenous leukemia (7,8). IRF-8 also regulates bone metabolism by suppressing osteoclast formation (9).

  1. Taniguchi, T. et al. (2001) Annu Rev Immunol 19, 623-55.
  2. Honda, K. and Taniguchi, T. (2006) Nat Rev Immunol 6, 644-58.
  3. Driggers, P.H. et al. (1990) Proc Natl Acad Sci U S A 87, 3743-7.
  4. Weisz, A. et al. (1992) J Biol Chem 267, 25589-96.
  5. Burchert, A. et al. (2004) Blood 103, 3480-9.
  6. Lehtonen, A. et al. (2003) Cytokine 24, 81-90.
  7. Holtschke, T. et al. (1996) Cell 87, 307-17.
  8. Fehr, T. et al. (1997) J Exp Med 185, 921-31.
  9. Zhao, B. et al. (2009) Nat Med 15, 1066-71.

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 BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq

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.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Revision 4
#5628

IRF-8 (D20D8) Rabbit mAb

Western Blotting Image 1: IRF-8 (D20D8) Rabbit mAb Expand Image
Western blot analysis of extracts from Raji and Jurkat cells, untreated or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml; overnight) using IRF-8 (D20D8) Rabbit mAb.
Western Blotting Image 2: IRF-8 (D20D8) Rabbit mAb Expand Image
Western blot analysis of extracts from THP-1, RL, and A20 cell lines using IRF-8 (D20D8) Rabbit mAb.
Western Blotting Image 3: IRF-8 (D20D8) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, mock transfected or transfected with human and mouse IRF-8 constructs, using IRF-8 (D20D8) Rabbit mAb.
Western Blotting Image 1: IRF-8 (D20D8) Rabbit mAb Expand Image
Simple WesternTM analysis of lysates (1.0 mg/mL) from THP-1 cells using IRF-8 (D20D8) Rabbit mAb #5628. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
No image available
Chromatin Immunoprecipitation Image 1: IRF-8 (D20D8) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from THP-1 and IRF-8 (D20D8) 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 GPR18/UBAC2, a known target gene of IRF8 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
Chromatin Immunoprecipitation Image 2: IRF-8 (D20D8) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from THP-1 and IRF-8 (D20D8) 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 13 (upper), including GPR18/UBAC2 (lower), a known target gene of IRF8 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: IRF-8 (D20D8) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 THP-1 and either IRF-8 (D20D8) 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 GPR18 promoter primers, SimpleChIP® Human MS4A7 Promoter Primers #9013, 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.