Cell Signaling Technology

Product Pathways - NF-kB Signaling

IRF-4 Antibody #4964

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC F ChIP H Endogenous 51 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  ChIP=Chromatin IP
Reactivity Key:  H=Human
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

IRF-4 Antibody detects endogenous levels of IRF-4 protein. The antibody does not cross-react with other family members at physiological levels.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues around Asp175 of human IRF-4. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from Karpas-620 (human) and A20 (mouse) cell lines using IRF-4 Antibody #4964 and IRF-4 (P173) Antibody #4948.

Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos, Raji, and HuT-78 cells, using IRF-4 Antibody.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of THP-1 cells (blue) and RPMI8226 cells (green) using IRF-4 Antibody.


IF-IC

IF-IC

Confocal immunofluorescent analysis of RPMI-8226 (left) and HeLa cells (right) using IRF-4 Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 H929 cells and either 20 μl of IRF-4 Antibody or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human SUB1 Promoter Primers #5156 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.

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, ISGF3γ/p48, 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-4 was independently cloned by three groups and demonstrated to have roles in different contexts of lymphoid regulation (3-5). First, IRF-4 (Pip) was found to associate with PU.1, a hematopoietic specific member of the ETS family, and to regulate the expression of B-cell specific genes (3). Second, it was characterized as a lymphoid-specific member of the IRF family (LSIRF) and able to bind to ISRE (4). Third, it was identified in activated T cells as a factor that binds to the promoter of the interleukin-5 gene (ICSAT), and shown to repress gene activation induced by IFN (5). IRF-4 is expressed in all stages of B cell development and in mature T cells, and is inducible in primary lymphocytes by antigen mimetic stimuli such as Concavalin A, CD3 crosslinking, anti-IgM and PMA treatment (4,5). Mice deficient in IRF-4 show normal distribution of B and T lymphocytes at 4 to 5 weeks, but later develop progressive generalized lymphadenopathy, suggesting a role for IRF-4 in the function and homeostasis of mature B- and T-lymphocytes (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. Eisenbeis, C.F. et al. (1995) Genes Dev 9, 1377-87.
  4. Matsuyama, T. et al. (1995) Nucleic Acids Res 23, 2127-36.
  5. Yamagata, T. et al. (1996) Mol Cell Biol 16, 1283-94.
  6. Mittrücker, H.W. et al. (1997) Science 275, 540-3.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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