Revision 4

#8478Store 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, IHC-Bond, IHC-P, IF-IC, FC-FP, C&R

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

45-48

Source/Isotype:

Rabbit IgG

UniProt ID:

#P10914

Entrez-Gene Id:

3659

Product Information

Product Usage Information

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
IHC Leica Bond 1:50
Immunohistochemistry (Paraffin) 1:100
Immunofluorescence (Immunocytochemistry) 1:200
Flow Cytometry (Fixed/Permeabilized) 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

IRF-1 (D5E4) XP® Rabbit mAb recognizes endogenous levels of total IRF-1 protein.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro261 of human IRF-1 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).

The IRF-1 transcription factor was originally identified as a regulator of virus-inducible enhancer-like elements of the IFN-β gene (3). IRF-1 is widely expressed and upregulated by viral infection or stimulation with IFN or other cytokines. IRF-1 is serine-phosphorylated by casein kinase II (CKII) at two clustered sites, one in the DNA-binding domain (amino acids 138-150) and another in the transactivation domain (amino acids 219-231) (4). Mutation analysis of the latter site suggests that these phosphorylation sites help regulate IRF-1 activity. Tyrosine phosphorylation has also been shown to be important in IFN-γ-mediated differentiation of myeloid cell lines (5). C-terminal SUMOylated IRF-1 inhibits apoptosis in tumor cells by repression of its transcriptional activity (6).

  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. Fujita, T. et al. (1988) EMBO J 7, 3397-405.
  4. Lin, R. and Hiscott, J. (1999) Mol Cell Biochem 191, 169-80.
  5. Kautz, B. et al. (2001) J Biol Chem 276, 37868-78.
  6. Park, J. et al. (2007) Proc Natl Acad Sci USA 104, 17028-33.

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 IP: Immunoprecipitation IHC-Bond: IHC Leica Bond IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) 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.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 4
#8478

IRF-1 (D5E4) XP® Rabbit mAb

Western Blotting Image 1: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Western blot analysis of HeLa cells, untreated or treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr), using IRF-1 (D5E4) XP® Rabbit mAb.
Western Blotting Image 2: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using IRF-1 (D5E4) XP® Rabbit mAb.
No image available
Immunohistochemistry Image 1: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse thymus using IRF-1 (D5E4) XP® Rabbit mAb performed on the Leica® BOND Rx.
Immunohistochemistry Image 1: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Imunohistochemical analysis of paraffin-embedded human breast carcinoma using IRF-1 (D5E4) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Immunohistochemistry Image 2: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Imunohistochemical analysis of paraffin-embedded human lymphoma using IRF-1 (D5E4) XP® Rabbit mAb.
Immunohistochemistry Image 3: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mosue thymus using IRF-1 (D5E4) XP® Rabbit mAb.
Immunohistochemistry Image 4: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Imunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or IFN gamma-treated (right), using IRF-1 (D5E4) XP® Rabbit mAb.
Immunofluorescence Image 1: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells, serum-starved (left) or treated with hIFNγ #8901 (100 ng/mL, 4 hr; right), using IRF-1 (D5E4) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin.
Flow Cytometry Image 1: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Human Interferon-γ (hIFN-γ) #8901 (green), using IRF-1 (D5E4) XP® Rabbit mAb.
CUT and RUN Image 1: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr) and IRF-1 (D5E4) XP® 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 ERAP2, a known target gene of IRF-1 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr) and IRF-1 (D5E4) XP® 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 figures show binding across chromosome 5 (upper), including ERAP2 (lower), a known target gene of IRF-1 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: IRF-1 (D5E4) XP® Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr) and either IRF-1 (D5E4) XP® 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 MOV10 intron 1 primers, human ERAP2 intron 1, 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.