IRF2BP2 Antibody

What is IRF2BP2 and why is it important for research?

IRF2BP2 is a transcriptional corepressor that belongs to a family of proteins characterized by an N-terminal zinc finger and a C-terminal RING domain. It plays crucial roles in regulating inflammatory pathways and has emerged as an important player in acute myeloid leukemia (AML) pathogenesis . IRF2BP2 was initially identified along with its homolog IRF2BP1 as a transcriptional corepressor for interferon regulatory factor 2 (IRF2) . Recent research has shown that IRF2BP2 interacts with the AP-1 heterodimer ATF7/JDP2 to regulate inflammatory gene expression . Additionally, IRF2BP2 has been found to repress lipolysis in adipocytes, which is essential for maintaining systemic metabolic homeostasis . The multifunctional nature of IRF2BP2 in various cellular processes makes its study critical for understanding both normal cellular function and disease mechanisms.

What are the key structural and molecular characteristics of IRF2BP2?

IRF2BP2 has a calculated and observed molecular weight of 61 kDa . Structurally, it contains an N-terminal zinc finger domain and a C-terminal RING domain . The zinc finger domain facilitates interactions with its homologs IRF2BP1 and IRF2BPL (also known as EAP1), allowing the formation of larger protein complexes . The RING domain has been implicated in ubiquitin ligase activity .

IRF2BP2's repressive function has been shown to be independent of histone deacetylases . Instead, E3 ubiquitin ligase activity and sumoylation mechanisms contribute to its repressive activity . The protein interacts with various transcription factors and chromatin regulators besides IRF2, including KLF2, NFAT1, ETO2, HNF4α, and TEAD4 .

How do IRF2BP2 antibodies differ from other transcription factor antibodies in terms of specificity and applications?

IRF2BP2 antibodies, such as the 18847-1-AP from Proteintech, are designed to specifically target the IRF2BP2 protein in various applications . Unlike antibodies against more common transcription factors, IRF2BP2 antibodies need to be carefully validated due to the protein's interactions with multiple partners and its homologs (IRF2BP1 and IRF2BPL) .

The specificity of IRF2BP2 antibodies is particularly important when studying its interactions with the AP-1 heterodimer ATF7/JDP2, as these interactions appear to be transient and context-dependent . When used in chromatin immunoprecipitation (ChIP) experiments, IRF2BP2 antibodies must be validated for specificity, as demonstrated in knockdown cells to ensure reliable results . For optimal research outcomes, validation across multiple applications (WB, IP, IHC, IF/ICC) is necessary, especially when investigating tissue-specific roles of IRF2BP2 in different experimental contexts .

What are the recommended dilutions and conditions for using IRF2BP2 antibody in various applications?

Based on empirical data, the following dilutions are recommended for IRF2BP2 antibody (18847-1-AP) in different applications:

It's crucial to note that these dilutions should be optimized for each experimental system to obtain optimal results . For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may alternatively be used .

For storage, the antibody should be kept at -20°C, where it remains stable for one year after shipment. The storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For the 20μl size, note that it contains 0.1% BSA .

How should researchers optimize ChIP-Seq protocols when using IRF2BP2 antibodies?

When conducting ChIP-Seq experiments with IRF2BP2 antibodies, several optimization steps are crucial:

• Antibody validation: Confirm the specificity of the IRF2BP2 antibody in knockdown or knockout cells before proceeding with ChIP-Seq, as demonstrated in published research .

• Cross-linking conditions: Since IRF2BP2 interacts with multiple proteins, including the transient binding with ATF7/JDP2 heterodimers, optimize formaldehyde cross-linking time (typically 10-15 minutes) to capture these interactions.

• Sonication parameters: Adjust sonication conditions to achieve chromatin fragments of 200-500bp for optimal resolution of IRF2BP2 binding sites.

• Control selection: Include appropriate controls such as IgG and input samples. Consider additional controls such as IRF2BP2 knockdown/knockout samples to identify specific binding events.

• Data analysis approach: When analyzing IRF2BP2 ChIP-Seq data, look for overlap with binding sites of known interaction partners, particularly ATF7 and JDP2. Studies have shown that approximately 80% of ATF7 peaks overlap with IRF2BP2 peaks in AML cells . Motif enrichment analysis should be performed to identify the binding motifs, which are likely to be similar to those of ATF7 .

• Integration with other data types: Combine ChIP-Seq data with RNA-Seq or other genomic datasets to correlate IRF2BP2 binding with transcriptional outcomes, particularly in genes involved in inflammatory pathways and myeloid cell function .

What cell lines and tissue samples have been validated for IRF2BP2 antibody applications?

IRF2BP2 antibody (18847-1-AP) has been validated in several cell lines and tissue samples:

For Western Blot applications, positive detection has been reported in:

• A549 cells

• HeLa cells

• K-562 cells

For Immunoprecipitation, positive detection has been validated in:

• K-562 cells

For Immunohistochemistry, positive staining has been observed in:

• Human lung squamous cell carcinoma tissue

• Human liver cancer tissue

For Immunofluorescence/ICC, positive signal has been detected in:

• HepG2 cells

In published research, the antibody has shown reactivity with both human and mouse samples . Additionally, IRF2BP2 antibodies have been successfully used in AML cell lines such as THP1 and MV4-11 for various applications, including ChIP-Seq experiments . The specificity of the antibody has been confirmed in IRF2BP2 knockdown cells .

What are common issues when detecting IRF2BP2 in Western blots and how can they be resolved?

When detecting IRF2BP2 in Western blots, researchers may encounter several challenges:

• Weak signal: Despite the high recommended dilution range (1:5000-1:50000) , some researchers may experience weak signals. To address this:

  • Decrease the antibody dilution

  • Increase protein loading (50-100 μg)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence detection systems

• Multiple bands: Since IRF2BP2 interacts with homologs IRF2BP1 and IRF2BPL , non-specific bands may appear. To improve specificity:

  • Increase blocking time or blocking agent concentration

  • Perform more stringent washing steps

  • Use validated positive controls like A549, HeLa, or K-562 cells

  • Consider using IRF2BP2 knockdown samples as negative controls

• Degradation products: IRF2BP2 may show degradation bands. To minimize this:

  • Add additional protease inhibitors to lysis buffer

  • Process samples quickly and maintain cold temperatures

  • Avoid repeated freeze-thaw cycles

• Inconsistent detection across cell types: Expression levels of IRF2BP2 vary across cell types. Optimize protein loading for each cell type and consider using cells with known high expression (like AML cell lines) as positive controls .

How can researchers differentiate between IRF2BP2 and its homologs (IRF2BP1 and IRF2BPL) in experimental settings?

Differentiating between IRF2BP2 and its homologs requires careful experimental design:

• Antibody selection: Choose antibodies raised against unique epitopes specific to IRF2BP2. The epitope information should be verified with the manufacturer.

• Western blot analysis: Although all three proteins possess both a zinc finger and a RING domain, they differ in molecular weight: IRF2BP2 is 61 kDa , while IRF2BP1 and IRF2BPL have different molecular weights. Run controls with recombinant proteins of each homolog.

• siRNA/shRNA validation: Perform knockdown experiments specific to each family member to confirm antibody specificity. For instance, IRF2BP2 knockdown should reduce the signal of the target band without affecting the bands corresponding to IRF2BP1 or IRF2BPL.

• Co-immunoprecipitation experiments: When performing IP experiments, validate the presence of IRF2BP2 versus its homologs using specific antibodies for each protein in the Western blot analysis of the immunoprecipitated material.

• Mass spectrometry: For definitive identification, consider using mass spectrometry analysis of immunoprecipitated samples to identify peptides unique to IRF2BP2 versus its homologs.

• RT-qPCR: Complement protein-level analyses with mRNA expression studies using primers specific to each family member.

What strategies can improve immunoprecipitation efficiency when studying IRF2BP2 interactions with partners like ATF7/JDP2?

To improve immunoprecipitation efficiency when studying IRF2BP2 interactions:

• Crosslinking optimization: Since interactions between IRF2BP2 and transcription factors like ATF7/JDP2 may be transient , consider using reversible crosslinking agents (such as DSP or formaldehyde) to stabilize these interactions.

• Buffer composition: Adjust lysis buffer conditions to preserve protein-protein interactions:

  • Use mild detergents (0.3-0.5% NP-40 or Triton X-100)

  • Include protease inhibitors and phosphatase inhibitors

  • Maintain physiological salt concentrations (120-150 mM NaCl)

  • Add 5-10% glycerol to stabilize protein complexes

• Antibody amount: Use the recommended 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . For detecting transient interactions, consider using the higher end of this range.

• Sequential IP approach: For complex interactions, consider a tandem IP approach where you first immunoprecipitate IRF2BP2 and then ATF7 or JDP2 from the eluted material (or vice versa).

• Validation controls: Include appropriate controls such as:

  • IgG control

  • Immunoprecipitation in IRF2BP2 knockdown cells

  • Reciprocal IP (using ATF7 or JDP2 antibodies)

• Protein complex elution: For studying multiple interaction partners, use gentle elution methods that preserve complex integrity rather than denaturing conditions.

How does IRF2BP2 binding pattern change in different disease contexts, particularly in AML?

Research indicates that IRF2BP2 binding patterns in disease contexts, particularly in AML, exhibit several important characteristics:

• Genomic occupancy in AML cells: ChIP-Seq experiments have identified over 21,500 significant IRF2BP2 binding sites in THP1 AML cells . These binding patterns show high consistency across different AML cell lines, including MV4-11 and PDX1601 , suggesting conserved regulatory mechanisms across AML subtypes.

• Co-localization with transcription factors: In AML cells, approximately 80% of ATF7 peaks overlap with IRF2BP2 peaks, indicating substantial co-occupancy . Further analysis revealed that about 90% of IRF2BP2 binding sites had some level of ATF7 binding , highlighting the importance of the IRF2BP2-ATF7/JDP2 regulatory axis in AML.

• Genomic distribution: IRF2BP2 is enriched at promoter regions in AML cells, similar to ATF7 . Motif enrichment analysis shows that IRF2BP2 binding sites possess motifs similar to those of ATF7 binding sites .

• Target gene functions: Gene ontology analysis reveals that the target genes of ATF7 and IRF2BP2 in AML cells are primarily associated with myeloid leukocyte activation and immune response , consistent with their role in regulating inflammatory pathways.

• Disease impact: IRF2BP2 is commonly upregulated in AML compared to normal cells , though its expression levels don't strongly correlate with patient survival, suggesting a complex role in disease progression .

• Functional consequences: Loss of IRF2BP2 in AML cells leads to overactivation of inflammatory pathways and strongly reduced proliferation , indicating that IRF2BP2 helps maintain a precise equilibrium between activating and repressive transcriptional mechanisms to create a pro-oncogenic inflammatory environment in AML cells .

What are the most effective genetic modification approaches for studying IRF2BP2 function?

When investigating IRF2BP2 function, researchers have employed several genetic modification strategies with varying effectiveness:

How can researchers effectively study the interplay between IRF2BP2 and inflammatory pathways?

To effectively study the interplay between IRF2BP2 and inflammatory pathways, researchers should consider these methodological approaches:

• Integrated genomic analyses: Combine ChIP-Seq data of IRF2BP2 with RNA-Seq analysis after IRF2BP2 knockdown to identify direct transcriptional targets involved in inflammatory signaling. This approach has revealed that IRF2BP2 regulates genes involved in myeloid leukocyte activation and immune response .

• Cytokine profiling: In IRF2BP2-depleted cells or tissues, measure changes in inflammatory cytokine and chemokine production using techniques such as:

  • Multiplex cytokine assays

  • ELISA

  • qPCR for inflammatory gene expression

  For example, in adipocyte-specific IRF2BP2 knockout mice, increased expression of inflammatory markers like Il1b, Il6, and Ccl2 has been observed .

• Flow cytometry analysis: Assess changes in inflammatory cell populations, as demonstrated in adipose tissue from IRF2BP2 knockout mice, which showed ~2.5-fold increase in proinflammatory F4/80+; CD11c+ cells .

• Transcription factor binding studies: Investigate how IRF2BP2 affects the binding and activity of inflammatory transcription factors, particularly focusing on the ATF7/JDP2 dimer. Co-immunoprecipitation and sequential ChIP (ChIP-reChIP) can reveal how IRF2BP2 modulates these factors' chromatin occupancy.

• Pathway inhibition experiments: Combine IRF2BP2 knockdown with inhibitors of specific inflammatory pathways (e.g., NF-κB, JAK-STAT, MAPK) to dissect which inflammatory cascades are directly affected by IRF2BP2.

• In vivo inflammation models: Utilize genetic models with altered IRF2BP2 expression in inflammation-related disease models to assess physiological relevance.

How should researchers interpret discrepancies between IRF2BP2 antibody results across different experimental platforms?

When facing discrepancies in IRF2BP2 antibody results across different experimental platforms, consider these interpretation guidelines:

• Application-specific differences: IRF2BP2 antibody performance varies across applications. For instance, an antibody might work well in Western blot (1:5000-1:50000 dilution) but require different optimization for IHC (1:500-1:2000) . Each application creates different epitope exposure conditions that affect antibody binding.

• Sample preparation variations: Different fixation methods (for IHC/IF) or lysis conditions (for WB/IP) can affect epitope accessibility. For IHC applications with IRF2BP2 antibody, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 is an alternative .

• Protein complex formation: IRF2BP2 exists in complexes with IRF2BP1 and IRF2BPL , and interacts with various partners like ATF7/JDP2 . These interactions may mask epitopes in specific cellular contexts or applications, leading to variable detection efficiency.

• Cell type and context-dependent modifications: Post-translational modifications or tissue-specific interaction partners may affect antibody recognition. For instance, IRF2BP2's function differs in AML cells versus adipocytes , which could influence detection patterns.

• Reconciliation strategy: When facing discrepancies:

  • Validate findings with multiple antibodies targeting different epitopes

  • Confirm specificity using genetic knockdown/knockout controls

  • Use recombinant protein standards where possible

  • Consider orthogonal detection methods (mass spectrometry)

  • Check if discrepancies correlate with known biological variables (cell activation state, tissue type)

What are the key considerations when analyzing IRF2BP2 ChIP-Seq data in relation to transcriptional outcomes?

When analyzing IRF2BP2 ChIP-Seq data to understand transcriptional outcomes, researchers should consider these critical factors:

• Integration with transcriptomic data: Correlate IRF2BP2 binding sites with gene expression changes (RNA-Seq) after IRF2BP2 knockdown to distinguish direct from indirect regulatory effects. Since IRF2BP2 functions as a repressor , genes with IRF2BP2 binding that show upregulation upon IRF2BP2 depletion are likely direct targets.

• Co-binding pattern analysis: Examine the relationship between IRF2BP2 binding and its partners, particularly ATF7 and JDP2. Research has shown that approximately 80% of ATF7 peaks overlap with IRF2BP2 peaks in AML cells , suggesting coordinated regulatory functions.

• Motif analysis interpretation: IRF2BP2 binding sites show enrichment for motifs similar to ATF7 binding sites , indicating that IRF2BP2 may be recruited to chromatin through specific transcription factors rather than binding DNA directly.

• Genomic distribution context: IRF2BP2 is enriched at promoters , suggesting a role in proximal gene regulation. Analyze whether different genomic contexts (promoters vs. enhancers) correlate with different regulatory outcomes.

• Pathway enrichment interpretation: Gene ontology analysis shows that IRF2BP2 target genes in AML cells are associated with myeloid leukocyte activation and immune response . This suggests that IRF2BP2 helps maintain a precise inflammatory balance in these cells.

• Kinetic considerations: Since IRF2BP2 interactions with some partners (like ATF7/JDP2) may be transient , consider how binding patterns may change under different cellular conditions or time points.

How can researchers distinguish between direct and indirect effects of IRF2BP2 in functional studies?

Distinguishing between direct and indirect effects of IRF2BP2 in functional studies requires a multi-faceted approach:

• Integrated genomic approaches:

  • Combine IRF2BP2 ChIP-Seq data with RNA-Seq after IRF2BP2 depletion

  • Genes that show both IRF2BP2 binding and expression changes are likely direct targets

  • Time-course RNA-Seq after acute IRF2BP2 depletion can help identify primary (early) versus secondary (late) effects

• Rescue experiments:

  • After IRF2BP2 knockdown, reintroduce wildtype IRF2BP2 or domain mutants

  • If a specific phenotype is reversed with wildtype but not with certain mutants, this can help map functional domains

  • For example, mutations in the RING domain, which has been implicated in ubiquitin ligase activity , might affect specific functions

• Targeted gene studies:

  • For putative direct targets identified in genomic screens, perform reporter assays with wildtype and mutated regulatory regions

  • This can confirm direct transcriptional regulation and identify critical cis-regulatory elements

• Protein-protein interaction mapping:

  • Use techniques like BioID or proximity ligation assays to identify proteins in close proximity to IRF2BP2

  • This helps build interaction networks and identify potential mediators of indirect effects

• Domain-specific perturbations:

  • Rather than depleting the entire protein, target specific domains or protein-protein interactions

  • For example, disrupting the IRF2BP2-ATF7/JDP2 interaction specifically would help distinguish effects mediated through this particular complex

• Context-specific analyses:

  • Compare IRF2BP2 functions across different cellular contexts (e.g., AML cells vs. adipocytes )

  • Conserved effects across contexts are more likely to represent core direct functions