IRF2BP1 Antibody

What is IRF2BP1 and what are its primary functions?

IRF2BP1 (Interferon Regulatory Factor 2 Binding Protein 1) is a nuclear protein that primarily functions as a transcriptional corepressor in an IRF2-dependent manner. Based on recent research, IRF2BP1 acts as a repressor without relying on histone deacetylase activities . It may also function as an E3 ubiquitin ligase towards JDP2, enhancing its polyubiquitination, and represses ATF2-dependent transcriptional activation . IRF2BP1 contains an N-terminal zinc finger and a C-terminal RING domain, characteristic features shared with its family members IRF2BP2 and IRF2BPL . Recent studies have also implicated IRF2BP1 in SUMOylation-dependent regulation of immediate early genes and cellular responses to growth factors .

What types of IRF2BP1 antibodies are available for research?

Several types of IRF2BP1 antibodies are available for research applications, as detailed in the table below:

When selecting an antibody, researchers should consider the specific application, target species, and the region of IRF2BP1 they wish to detect.

How can I optimize Western blot protocols for detecting IRF2BP1 in different cell types?

Optimizing Western blot protocols for IRF2BP1 detection requires attention to several key factors:

• Sample preparation: For cell lines like Jurkat, HepG2, and MOLT-4, both whole cell lysates (30 μg) and nuclear extracts (10 μg) have been successfully used . Nuclear extracts may provide enriched IRF2BP1 signal due to its predominant nuclear localization.

• Antibody selection and dilution:

  • For sheep polyclonal antibodies (e.g., AF6800), 1 μg/mL has been effective

  • For mouse monoclonal antibodies (e.g., ab236392), 1/2000 dilution has shown good results

  • For rabbit polyclonal antibodies (e.g., 13698-1-AP), 1:500-1:1000 dilution is recommended

• Detection system: HRP-conjugated secondary antibodies specific to your primary antibody's host species are essential. For example, when using sheep primary antibodies, HRP-conjugated Anti-Sheep IgG Secondary Antibody has been successful .

• Expected band size: IRF2BP1 is typically detected at approximately 62-72 kDa , though some sources report detection at around 70 kDa .

• Controls: Consider using cell lines with known IRF2BP1 expression (HeLa, HEK-293, Jurkat) as positive controls . Treatment with TGF-beta 1 (10 ng/mL for 2 hours) has been shown to affect IRF2BP1 levels and can serve as an experimental condition .

What are the considerations for designing ChIP experiments to study IRF2BP1's role in transcriptional regulation?

ChIP (Chromatin Immunoprecipitation) experiments to study IRF2BP1's role in transcriptional regulation should consider:

• Antibody selection: Choose ChIP-validated antibodies for IRF2BP1. The polyclonal antibody 13698-1-AP has been cited for ChIP applications .

• Crosslinking conditions: Standard formaldehyde crosslinking (1% for 10 minutes at room temperature) is generally appropriate for transcription factors and corepressors.

• Target genes: Focus on genes known to be regulated by IRF2 or ATF2, as IRF2BP1 functions as a corepressor for these transcription factors . Additionally, immediate early genes such as DUSP1, ATF3, Egr2, and Fos have been identified as targets of IRF2BP1-mediated regulation .

• Controls:

  • Input control (non-immunoprecipitated chromatin)

  • Negative control (IgG from the same species as the IRF2BP1 antibody)

  • Positive control (antibody against a known transcription factor that binds the same regions)

• Experimental conditions: Consider examining IRF2BP1 binding under different conditions, such as with and without growth factor stimulation (e.g., EGF), as IRF2BP1's SUMOylation status and its binding to target genes have been shown to change in response to such stimuli .

How can I investigate the relationship between IRF2BP1 SUMOylation status and its function?

Investigating the relationship between IRF2BP1 SUMOylation status and its function requires a multi-faceted approach:

• Generation of SUMOylation-deficient mutants: Create IRF2BP1 mutants where key lysine residues are mutated to arginine. Research has identified specific variants (e.g., the V578A variant) that are SUMOylation-deficient .

• Cell line models: Establish cell lines expressing wild-type IRF2BP1 versus SUMOylation-deficient mutants after depleting endogenous IRF2BP1. This approach has been used to study the differential effects on gene expression .

• Stimulation experiments: Compare the effects of growth factor stimulation (e.g., EGF) on wild-type versus SUMOylation-deficient IRF2BP1. Research has shown that EGF stimulation leads to transient deSUMOylation of IRF2BP1, affecting the expression of immediate early genes .

• Transcriptomic analysis: Use microarrays or RNA-seq to compare gene expression profiles between wild-type and SUMOylation-deficient IRF2BP1-expressing cells, both in steady state and after stimulation .

• ChIP-seq: Map genome-wide binding sites of wild-type versus SUMOylation-deficient IRF2BP1, and correlate with gene expression changes.

Why might I observe multiple bands when detecting IRF2BP1 by Western blot?

Multiple bands in IRF2BP1 Western blots can occur for several reasons:

• Post-translational modifications: IRF2BP1 undergoes SUMOylation , which can cause shifts in molecular weight. The deSUMOylated and SUMOylated forms may appear as distinct bands.

• Isoforms: While not explicitly mentioned in the search results, many proteins have multiple isoforms due to alternative splicing.

• Proteolytic degradation: Sample preparation without adequate protease inhibitors can lead to degradation products.

• Cross-reactivity: Some antibodies may cross-react with related proteins like IRF2BP2 or IRF2BPL, which share homologous domains .

• Experimental variables: Different cell types or treatments can affect IRF2BP1 expression and modification. For example, TGF-beta 1 treatment has been shown to affect IRF2BP1 levels in certain cell lines .

To address this issue:

• Compare your results with published Western blots. For instance, IRF2BP1 has been reported at approximately 70 kDa in Jurkat, HepG2, and MOLT-4 cell lines .

• Use positive controls like HeLa, HEK-293, or Jurkat cells that have been validated for IRF2BP1 expression .

• Consider performing immunoprecipitation followed by Western blot to enrich for IRF2BP1 and confirm specificity.

How can I reconcile contradictory data about IRF2BP1 function in different cell types or experimental conditions?

Reconciling contradictory data about IRF2BP1 function requires systematic analysis and careful consideration of several factors:

• Cell type-specific effects: IRF2BP1 may interact with different partners and regulate different genes depending on the cellular context. For example, the IRF2BP family has been implicated in different roles in immune cells versus other cell types .

• Experimental conditions: Growth factors like EGF can trigger transient deSUMOylation of IRF2BP1, changing its function . Different studies may use different stimulation protocols.

• Protein complexes: IRF2BP1 functions within protein complexes, including with IRF2BP2 and IRF2BPL . The composition of these complexes may vary across cell types.

• Post-translational modifications: SUMOylation status affects IRF2BP1 function . Different studies may be capturing IRF2BP1 in different modification states.

To reconcile contradictory data:

• Directly compare experimental conditions, including cell types, treatments, time points, and assay methods.

• Perform parallel experiments with multiple cell types under identical conditions.

• Consider the activation state of signaling pathways that might affect IRF2BP1 function.

• Validate findings using complementary approaches (e.g., both gain- and loss-of-function studies).

What controls should I include when performing immunoprecipitation with IRF2BP1 antibodies?

When performing immunoprecipitation (IP) with IRF2BP1 antibodies, include these essential controls:

• Input control: 5-10% of the pre-IP lysate to verify the presence of IRF2BP1 in starting material.

• Negative controls:

  • IgG control: Use the same amount of non-specific IgG from the same species as your IRF2BP1 antibody

  • Beads-only control: Include a sample with beads but no antibody

  • Lysate from cells where IRF2BP1 has been knocked down (if available)

• Positive controls:

  • Lysate from cells overexpressing IRF2BP1 (if available)

  • Cell lines with known IRF2BP1 expression (e.g., HEK-293 cells, which have been validated for IRF2BP1 IP)

• Reciprocal IP: If studying interaction partners, perform IP with antibodies against the suspected interacting protein and blot for IRF2BP1.

For IP of IRF2BP1, the recommended antibody dilution for polyclonal antibodies like 13698-1-AP is 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate .

What are the key differences between using monoclonal versus polyclonal antibodies for IRF2BP1 detection?

The choice between monoclonal and polyclonal antibodies for IRF2BP1 detection involves several important considerations:

Monoclonal Antibodies (e.g., OTI3A6 ):

• Specificity: Generally highly specific for a single epitope, which can reduce background but may be sensitive to epitope masking due to protein modifications or conformation.

• Consistency: Provide high batch-to-batch reproducibility, making them ideal for long-term studies.

• Applications: The monoclonal antibody OTI3A6 has been validated for Western blot and IHC-P applications .

• Epitope recognition: Mouse monoclonal antibody ab236392 recognizes a specific region within human IRF2BP1 aa 450 to C-terminus .

Polyclonal Antibodies (e.g., AF6800, 13698-1-AP):

• Epitope coverage: Recognize multiple epitopes, increasing detection sensitivity and resilience to partial denaturation or modification.

• Applications: Often validated for multiple techniques. For example, rabbit polyclonal 13698-1-AP is validated for WB, IP, IHC, IF/ICC, ChIP, and ELISA .

• Species reactivity: Many polyclonal antibodies show cross-reactivity with multiple species due to recognizing conserved epitopes. For example, some polyclonal antibodies react with human, mouse, and rat IRF2BP1 .

• Batch variation: May show more batch-to-batch variation compared to monoclonals.

How can I determine the specificity of an IRF2BP1 antibody for my experimental system?

Determining IRF2BP1 antibody specificity for your experimental system involves several validation steps:

• Western blot validation:

  • Compare with positive controls like HeLa, HEK-293, Jurkat, MOLT-4, or HepG2 cells where IRF2BP1 expression has been verified .

  • Check if the detected band matches the expected molecular weight (approximately 62-72 kDa) .

  • Perform knockdown experiments (siRNA or shRNA against IRF2BP1) and verify reduced signal.

  • If possible, use IRF2BP1-overexpressing cells as additional positive controls.

• Cross-reactivity assessment:

  • Test the antibody in systems where related proteins (IRF2BP2, IRF2BPL) are differentially expressed to assess cross-reactivity.

  • Consider that some antibodies cross-react with IRF2BP1 homologs in other species due to conserved epitopes.

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with your IRF2BP1 antibody and analyze precipitated proteins by mass spectrometry.

  • This identifies the specific protein(s) recognized by your antibody.

• Immunofluorescence specificity:

  • Confirm nuclear and/or cytoplasmic localization pattern consistent with IRF2BP1's known distribution .

  • Perform parallel experiments with knockdown cells to confirm specificity of staining.

How is IRF2BP1 implicated in the regulation of inflammation and immune responses?

While IRF2BP1 itself has limited direct evidence linking it to inflammation in the current literature, its family member IRF2BP2 has well-established roles in inflammatory regulation, and their functional similarities suggest potential parallel roles for IRF2BP1:

• Transcriptional regulation: IRF2BP1, like IRF2BP2, functions as a transcriptional corepressor in an IRF2-dependent manner . IRF2 is involved in regulating type I interferon responses, suggesting a potential role for IRF2BP1 in modulating inflammation through this pathway.

• Connection to AP-1 transcription factors: IRF2BP1 represses ATF2-dependent transcriptional activation . The AP-1 family of transcription factors, including ATF2, regulate many inflammatory genes.

• Family member functions: IRF2BP2 has been shown to:

  • Suppress inflammation in macrophages and microglia

  • Inhibit the expression of programmed death ligand 1 (PD-L1)

  • Interact with and counteract the AP-1 heterodimer ATF7/JDP2, which is involved in activating inflammatory pathways in acute myeloid leukemia cells

• IRF2BP1 in gene expression: Microarray analysis revealed that IRF2BP1 regulates genes involved in cell adhesion, proliferation, and response to growth factor stimuli , processes that can influence inflammatory responses.

What is the significance of IRF2BP1's potential E3 ligase activity in cellular signaling?

IRF2BP1's potential E3 ubiquitin ligase activity has significant implications for cellular signaling:

• Molecular basis: IRF2BP1 contains a C-terminal RING domain , a characteristic feature of many E3 ubiquitin ligases, and is described as a "Probable RING-type E3 ubiquitin transferase" .

• Substrate specificity: IRF2BP1 may act as an E3 ligase towards JDP2, enhancing its polyubiquitination . JDP2 (Jun dimerization protein 2) is a member of the AP-1 transcription factor family involved in various cellular processes including stress responses and transformation.

• Signaling regulation mechanisms:

  • Protein turnover: By promoting ubiquitination, IRF2BP1 may regulate the stability and abundance of target proteins like JDP2.

  • Activity modulation: Ubiquitination can alter protein activity or localization without degradation.

  • Protein-protein interactions: Ubiquitin modifications can create or disrupt binding interfaces.

• Integration with transcriptional repression: IRF2BP1's dual roles as a transcriptional corepressor and potential E3 ligase suggest it may coordinate gene expression regulation at multiple levels:

  • Direct transcriptional repression through IRF2-dependent mechanisms

  • Post-translational control of transcription factors through ubiquitination

How might IRF2BP1 SUMOylation status contribute to cancer progression or therapy response?

The SUMOylation status of IRF2BP1 could significantly impact cancer progression and therapy response through several mechanisms:

• Regulation of immediate early genes: SUMOylation of IRF2BP1 affects the expression of immediate early genes (IEGs) like DUSP1, ATF3, Egr2, and Fos . These genes regulate cellular responses to growth factors and stress, with implications for:

  • Cell proliferation and survival

  • Resistance to apoptosis

  • Response to therapy-induced stress

• Growth factor signaling: Transient deSUMOylation of IRF2BP1 occurs in response to EGF stimulation . Aberrant growth factor signaling is a hallmark of many cancers, suggesting IRF2BP1 SUMOylation could modulate oncogenic signaling pathways.

• Gene expression patterns: Microarray analysis revealed that IRF2BP1 SUMOylation status affects genes involved in cell adhesion, proliferation, and growth factor responses , all processes relevant to cancer progression and metastasis.

• Connection to MAPK signaling: IRF2BP1 regulates DUSP1 , a phosphatase that inhibits MAPK signaling. Many cancer therapies target the MAPK pathway, suggesting IRF2BP1 SUMOylation could influence therapy response.

• Family member connections: IRF2BP2, a homolog of IRF2BP1, has been identified as a tumor suppressor in hepatocellular carcinoma and regulates the Hippo pathway . IRF2BP2 also inhibits PD-L1 expression , suggesting potential roles in cancer immune evasion.