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.

限制使用

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Revision 4
#5628

IRF-8 (D20D8) Rabbit mAb

Western Blotting Image 1: IRF-8 (D20D8) Rabbit mAb Expand Image
使用 IRF-8 (D20D8) Rabbit mAb 对未处理或经 Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml; 过夜)处理的 Raji 和 Jurkat 细胞的提取物进行蛋白质印迹分析。
Western Blotting Image 2: IRF-8 (D20D8) Rabbit mAb Expand Image
使用 IRF-8 (D20D8) Rabbit mAb 对 THP-1、RL7 和 A20 细胞系的提取物进行蛋白质印迹分析。
Western Blotting Image 3: IRF-8 (D20D8) Rabbit mAb Expand Image
使用 IRF-8 (D20D8) Rabbit mAb 对转染空载或转染表达人或小鼠的 IRF-8 载体的 293T 细胞提取物进行蛋白质印迹分析。
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. 虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。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.
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Chromatin Immunoprecipitation Image 1: IRF-8 (D20D8) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,以取自于THP-1 细胞的交联染色质和 IRF-8 (D20D8) Rabbit mAb 进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。图形显示在 GPR18/UBAC2 内结合,GPR18/UBAC2 是一种已知的 IRF8 靶标基因(参见包含 ChIP-qPCR 数据的其他图形)。如需了解其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: IRF-8 (D20D8) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,以取自于THP-1 细胞的交联染色质和 IRF-8 (D20D8) Rabbit mAb 进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体13(上图)内的结合,包括 IRF8 的已知靶基因 GPR18/UBAC2(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: IRF-8 (D20D8) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对 4 x 106 THP-1 的交联染色质和 IRF-8 (D20D8) Rabbit mAb 或 Normal Rabbit IgG #2729 进行染色质免疫沉淀。使用人类 GPR18 启动子引物 、SimpleChIP® Human MS4A7 Promoter Primers #9013 和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 法对富集的 DNA 进行定量。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。