Invitrogen

IRF4 Monoclonal Antibody (3E4), eBioscience™

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This Antibody was verified by Relative expression to ensure that the antibody binds to the antigen stated. View Details

20 Published Figures

19 References

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IRF4 Antibody in Western Blot (WB) — Western blot was performed using Anti-IRF4 Monoclonal Antibody (3E4) (Product # 14-9858-82) and a 51kDa band corresponding to IRF4 was observed in IM9 and was absent in MOLT-4, Jurkat and U-937 as reported. Whole cell extracts (30 µg lysate) of IM9 (Lane 1), MOLT-4 (Lane 2), Jurkat (Lane 3) and U-937 (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (5ug/ml) and detected by chemiluminescence F(ab)2-Rabbit anti-Rat IgG (H+L) Secondary Antibody, HRP (Product # PA1-29927; 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

IRF4 Antibody — Antibody specificity was demonstrated by detection of differential basal expression of the target across cell lines tested owing to their inherent genetic constitution. Relative expression of IRF4 was observed in IM9 in comparison to MOLT-4, Jurkat and U-937 using Anti-IRF4 Monoclonal antibody (3E4) (Product # 14-9858-82) in Western Blot. {RE}

IRF4 Antibody in Immunohistochemistry (IHC) — Published figure using IRF4 monoclonal antibody (Product # 14-9858-82) in Immunohistochemistry

IRF4 Antibody in Flow Cytometry (Flow) — Fig. 2 Decreased number of plasma cells in Ufbp1 F/F CD19 cre mice. a Bone marrow cells from indicated mice were stained with BLIMP1, CD138, IRF4, and Pax5, and analyzed by flow cytometry. Shown is the BLIMP1 versus CD138 (upper panel), and IRF4 versus Pax5 staining (lower panel) b Enumeration of frequencies of BLIMP1 + CD138 + and IRF4 + Pax5 - cells in a ( n = 3 mice/genotype). c Ufbp1 F/F and Ufbp1 F/F CD19 cre mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to keyhole limpet hemocyanin (NP-KLH) as in Fig. 1c were analyzed for plasma cells (BLIMP1 + CD138 + and IRF4 + Pax5 - ) in bone marrow by flow cytometry at the indicated time points after immunization. d Enumeration of the frequencies of plasma cells (BLIMP1 + CD138 + and IRF4 + Pax5 - ) in c ( n = 3 mice/genotype). e Indicated mice were immunized as in Fig. 1c . Twenty-one days later, bone marrow cells were analyzed for the presence of NP-specific IgG1 + ASCs by ELISpot. Shown is a photograph of representative ELISpot wells. f Number (mean +- SEM) of NP-specific IgG1 + ELISpots in e ( n = 6 mice/genotype). g Ufbp1 F/F and Ufbp1 F/F CD19 cre mice were immunized with NP-KLH in Sigma Adjuvant System and on day 10, 7-AAD - CD4 - CD8 - Gr-1 - CD11b - splenic cells were analyzed for IgD and NP staining by flow cytometry. h Enumeration of the frequency of IgD - NP + cells in g ( n = 3 mice/genotype). i BLIMP1 and CD138 expression by IgD - NP + cells in g was an

IRF4 Antibody in ChIP assay (ChIP) — Figure 3. Increased Binding of IRF4 at the Il17 Locus and IRF4 and RORgammat Requirement for IL-17A Production in Blimp-1 -/- Foxp3 + Treg (A) Representation of the Il17 locus showing RORgammat and IRF4 consensus binding sites (top) and RORgt ChIP (bottom) in Blimp-1 +/+ and Blimp-1 -/- CD4 + Foxp3 GFP+ Treg and wild-type pTh17 cells. (B) IL-17A expression in TCRbeta + CD4 + Foxp3 + Treg (MLNs) from Prdm1 +/+ Rorc +/+ Foxp3 CREYFP+ (Ctrl), Prdm1 F/F Rorc +/+ Foxp 3CREYFP+ (knockout [KO]), and Prdm1 F/F Rorc F/F Foxp3 CREYFP+ (double knockout [DKO]) mice and expression of Foxp3 , Prdm1 , Rorc , and Il17a mRNA (bottom graphs) in sorted and in vitro -stimulated (phorbol myristate acetate [PMA] and ionomycin for 4 hr) CD4 + Foxp3 CREYFP+ cells. (C) IRF4 ChIP at the Il17 CNS7 and Il10 Intron I regions in Blimp1 +/+ and Blimp1 -/- CD4 + Foxp3 GFP+ Treg and wild-type pTh17 cells. (D) Irf4 , Il17a , and Il10 mRNA expression following Irf4 siRNA knockdown (SC, scrambled control). Data shown are representative of at least two independent experiments. Error bars show SEM (n >= 3 per group). *p < 0.05 and **p < 0.01, one-way ANOVA.

IRF4 Antibody in ELISA (ELISA) — Figure 2. SphK1 -/- T Cells Secrete Both IFN-gamma and IL-17 upon TCR Stimulation (A-C) pMel and pMel-SphK -/- T cells activated for 3 days with cognate antigen gp100 were antigen-restimulated overnight, and the supernatant was used for ELISA-based determination of secreted cytokine: (A) IFN-gamma, (B) IL-17, (C) RORgammat and IRF4 levels. (D) Splenic T cells from IL-17-GFP reporter or SphK1 -/- -IL-17-GFP mice were cultured in Th17 polarizing condition for 3 days, restimulated with anti-CD3 (2 ug/mL) and anti-CD28 antibody (5 ug/mL) for 4 h, and the percentage of IL-17-GFP-positive cells were determined by flow cytometry. (E) Three-day TCR-activated WT and SphK1 -/- T cells were analyzed for expression of stemness-associated genes using mRNA. Data are representative of three independent experiments in (A)-(E). (F) C57BL/6 mice (n = 10 mice/group) were inoculated subcutaneously (s.c.) with 0.3 x 10 6 B16-F10 melanoma cells for 9 days, after which mice were either kept untreated or adoptively transferred, either with 1 x 10 6 pMel or pMel-SphK1 -/- T cells. Tumor growth was measured using digital calipers twice weekly. Left panel: estimated growth curves for each experimental condition. Right panel: survival curve (p value pMel versus pMel SphK1 -/- is 1e-05). (G) Upper panel: C57BL/6 mice (n = 5 mice/group) were inoculated (s.c.) with 0.25 x 10 6 B16-F10 melanoma cells for 14 days, then 1 x 10 6 3 days gp100-activated pMel T cells, pMel SphK1 -/- T cells, or pMel T cells trea

Product Details

Applications

Tested Dilution

Publications

Western Blot (WB)

5 µg/mL

View 1 publication 1 publication

Immunohistochemistry (IHC)

View 1 publication 1 publication

Flow Cytometry (Flow)

View 15 publications 15 publications

ELISA (ELISA)

View 1 publication 1 publication

ChIP assay (ChIP)

View 1 publication 1 publication

Product Specifications

Species Reactivity

Human, Mouse

Published species

Human, Mouse

Host/Isotype

Rat / IgG1, kappa

Class

Monoclonal

Type

Antibody

Clone

3E4

Conjugate

Unconjugated

Form

Liquid

Concentration

0.5 mg/mL

Purification

Affinity chromatography

Storage buffer

PBS, pH 7.2

Contains

0.09% sodium azide

Storage conditions

4° C

RRID

AB_10804654

Product Specific Information

Description: The monoclonal antibody 3E4 reacts with human and mouse interferon regulatory factor 4 (IRF4). IRF4 is a 52 kDa transcription factor with roles in B cell, T cell and macrophage function. In B cells, IRF4 is highly expressed in mature plasma cells and plays a crucial role in their differentiation. IRF4 has been shown to interact with PU.1 and control the transcription of many B cell-specific genes including Prdm1, which encodes Blimp1. In T cells, IRF4 has been implicated in regulatory T (Treg), T helper (Th)2, Th9 and Th17 cell development and function. This transcription factor is upregulated upon T cell activation and is expressed in mature T cells. Studies have shown that IRF4 directly induces Blimp1 expression in Tregs, leading to IL-10 expression. IRF4 has also been demonstrated to be involved in macrophage polarization and regulation. Lastly, in addition to its roles in normal immune function, IRF4 expression has been reported to be upregulated in many blood-related cancers.

Applications Reported: This 3E4 antibody has been reported for use in western blotting.

Applications Tested: This 3E4 antibody has been tested by western blot of reduced cell lysate prepared from splenocytes cultured for 3 days in the presence of plate-bound Anti-Mouse CD3 and soluble Anti-Mouse CD28. This antibody can be used at less than or equal to 5 µg/mL. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

Purity: Greater than 90%, as determined by SDS-PAGE.

Aggregation: Less than 10%, as determined by HPLC.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

The IRF (interferon regulatory factor) family of transcription factors are characterized by an unique tryptophan pentad repeat DNA-binding domain. The IRFs are important in the regulation of interferons in response to infection by virus, and in the regulation of interferon-inducible genes. IRF4 is lymphocyte specific and negatively regulates toll-like-receptor (TLR) signaling that is central to the activation of innate and adaptive immune systems. A chromosomal translocation involving this gene and the IgH locus, t(6;14)(p25;q32), may be a cause of multiple myeloma. Alternatively spliced transcript variants have been found.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: Interferon regulatory factor 4; IRF-4; LSIRF; Lymphocyte-specific interferon regulatory factor; Multiple myeloma oncogene 1; NF-EM5; PU.1 interaction partner; Sfpi1/PU.1 interaction partner; Transcriptional activator PIP

Gene Aliases: AI385587; IRF-4; IRF4; LSIRF; MUM1; NF-EM5; SHEP8; Spip

UniProt ID: (Human) Q15306, (Mouse) Q64287

Entrez Gene ID: (Human) 3662, (Mouse) 16364

Molecular Function: winged helix/forkhead transcription factor