n4bp1 Antibody

What is N4BP1 and why is it important to detect it using antibodies?

N4BP1 is a multifunctional protein that serves as a critical regulator of several cellular processes. It was initially identified as a binding partner of the E3 ubiquitin ligase NEDD4 . Recent research has revealed its significance as:

• A potent suppressor of proinflammatory cytokine production that regulates innate immune signaling

• A novel linear ubiquitin reader that negatively regulates NFκB signaling via a unique dimerization-dependent ubiquitin-binding module (LUBIN)

• A regulator of the Notch signaling pathway involved in cortical development

• An endoribonuclease that degrades specific mRNA substrates

Detecting N4BP1 using antibodies is essential for understanding its role in these diverse biological processes and how its dysregulation may contribute to inflammatory and immune disorders.

What are the common applications for N4BP1 antibodies in research?

N4BP1 antibodies are valuable tools for investigating multiple aspects of this protein's function through various experimental approaches:

What should researchers know about N4BP1 protein structure when selecting antibodies?

Understanding N4BP1's domain architecture is crucial for antibody selection and experimental design:

N4BP1 contains several functional domains that mediate its diverse activities :

• Two RNA-binding KH domains in the N-terminal region

• Two ubiquitin-binding domains: a UBA-like (Ubiquitin Associated-like) domain and a CUE-like (Coupling of Ubiquitin conjugation to ER degradation-like) domain (also called CoCUN)

• An NYN (N4BP1, YacP-like Nuclease) ribonuclease domain in the C-terminal region

When selecting antibodies, researchers should consider which domain they wish to detect, especially if studying specific functions of N4BP1. For example, antibodies targeting the KH domains would be relevant for studying RNA-binding activities, while those targeting the ubiquitin-binding domains would be useful for investigating interactions with ubiquitin chains .

What are the optimal conditions for using N4BP1 antibodies in Western blotting?

For effective Western blotting of N4BP1:

• Sample Preparation:

  • Use strong lysis buffers containing 1% SDS or guanidinium chloride for complete extraction, as N4BP1 can be tightly associated with nucleolar structures

  • Include protease inhibitors to prevent degradation

  • For studying ubiquitination, add deubiquitinase inhibitors (e.g., N-ethylmaleimide)

• Gel Electrophoresis:

  • Use 6-8% gels to resolve the 130 kDa N4BP1 protein effectively

  • When studying polyubiquitination, use gradient gels (4-15%) to visualize high molecular weight smears

• Transfer and Detection:

  • Perform wet transfer for large proteins like N4BP1

  • Block with 5% BSA rather than milk to reduce background

  • Incubate with primary antibody at 1:1000 dilution overnight at 4°C

  • Include positive controls such as cell lines known to express N4BP1

• Special Considerations:

  • Be aware that N4BP1 may appear as multiple bands due to post-translational modifications

  • When studying caspase-mediated cleavage, look for specific fragment sizes resulting from cleavage after D424 and D490 residues

How can I optimize immunoprecipitation protocols with N4BP1 antibodies?

For successful immunoprecipitation of N4BP1 and its interaction partners:

• Lysis Conditions:

  • Use NP-40 or RIPA buffer for standard IP

  • For studying transient interactions, consider crosslinking with formaldehyde or DSP

  • Include phosphatase inhibitors in addition to protease inhibitors to preserve phosphorylation-dependent interactions

• Immunoprecipitation Procedure:

  • Use 1:100 dilution of antibody for immunoprecipitation

  • Pre-clear lysates to reduce non-specific binding

  • Incubate with antibody overnight at 4°C with gentle rotation

  • For co-IP studies of N4BP1 with NICD (Notch intracellular domain), gentle washing conditions are critical to preserve interactions

• Verification of Results:

  • Include IgG controls to assess non-specific binding

  • Perform reverse IP when studying novel interactions

  • For difficult-to-detect interactions, consider proximity ligation assays as an alternative approach

What methods should be used to study N4BP1 ubiquitination patterns?

N4BP1 undergoes both mono- and poly-ubiquitination, which are critical for its function and regulation . To study these modifications:

• In vivo Ubiquitination Assays:

  • Co-transfect cells with N4BP1 and tagged ubiquitin (HA-Ub or Myc-Ub)

  • Treat with proteasome inhibitors (MG132) to preserve ubiquitinated species

  • Perform denaturing IP (with 1% SDS or 6M guanidinium chloride) to disrupt non-covalent interactions

  • Analyze by Western blot, looking for both mono-ubiquitination (discrete band at ~120 kDa) and poly-ubiquitination (high molecular weight smear)

• Domain-specific Analysis:

  • Use N4BP1 mutants lacking specific domains (UBA-like or CUE-like) to determine their role in ubiquitin binding

  • The FP motif in the N4BP1 CoCUN domain is critical for recognizing the canonical hydrophobic patch in ubiquitin

• Ubiquitin Chain Specificity:

  • Use chain-specific ubiquitin antibodies to determine whether N4BP1 is modified by K48, K63, or linear ubiquitin chains

  • For studying linear ubiquitin binding, utilize the unique LUBIN domain of N4BP1

How can I investigate the role of N4BP1 in regulating NF-κB signaling?

N4BP1 negatively regulates NF-κB signaling by binding to NEMO and inhibiting its dimerization or oligomerization . To study this:

• Signaling Pathway Analysis:

  • Monitor NF-κB activation using luciferase reporter assays in the presence or absence of N4BP1

  • Compare TLR-dependent activation with and without N4BP1 expression

  • Focus specifically on TRIF-independent (TLR2, TLR7, or TLR9-mediated) NF-κB activation, which is enhanced in N4BP1-deficient cells

• Protein-Protein Interaction Studies:

  • Examine the interaction between N4BP1 and NEMO using co-IP

  • Focus on the interaction between N4BP1's UBA-like and CUE-like domains with the NEMO COZI domain

  • Use FRET or BiFC to visualize these interactions in living cells

• Caspase-8 Regulation:

  • Study how TRIF activation leads to caspase-8-mediated cleavage of N4BP1 after residues D424 and D490

  • Use caspase inhibitors or non-cleavable N4BP1 mutants to assess the functional consequences of this regulation

  • Monitor cytokine production as a readout of pathway activation

What approaches can detect N4BP1's role in mRNA degradation?

N4BP1 functions as an endoribonuclease that degrades specific mRNA targets . To investigate this activity:

• Target Identification:

  • Perform RNA immunoprecipitation (RIP) with N4BP1 antibodies to identify bound mRNAs

  • Conduct transcriptome analysis comparing wild-type and N4BP1-deficient cells

  • Focus on known targets like Fos-C and Jun-B transcripts

• Degradation Assays:

  • Use RNA stability assays with actinomycin D treatment to measure half-lives of target mRNAs

  • Perform in vitro RNA degradation assays with purified N4BP1 and synthetic RNA substrates

  • Compare wild-type N4BP1 with mutants (particularly G93D and D623N) that lack endoribonuclease activity

• Domain Requirement Analysis:

  • Employ domain deletion mutants to determine which domains are necessary for RNA binding and degradation

  • The KH domain is required but not sufficient for N4BP1's inhibitory function on mRNA targets

  • Both RNA-binding activity of the KH domain and endoribonuclease activity are essential for N4BP1 function

How can I study N4BP1's role in Notch signaling regulation?

N4BP1 negatively regulates Notch signaling by promoting NICD protein turnover through the ubiquitin-proteasome pathway . To investigate this:

• Protein Stability Assays:

  • Perform cycloheximide chase experiments to evaluate NICD protein stability in the presence or absence of N4BP1

  • Use proteasome inhibitors (MG132) and lysosomal inhibitors (chloroquine) to determine the degradation pathway

  • Compare effects on full-length Notch1 versus the NICD fragment

• Ubiquitination Analysis:

  • Co-express HA-Ub with NICD and N4BP1 to detect ubiquitin conjugation

  • Use the CoCUN domain mutants of N4BP1 to assess the role of this domain in NICD ubiquitination

  • Monitor formation of high-molecular-weight ubiquitin smears after anti-Flag immunoprecipitation

• Functional Readouts:

  • Measure Notch pathway activity using luciferase reporter assays (4×CBF1-Luc)

  • Analyze expression of Notch target genes (Hey1, BLBP) by qRT-PCR

  • Use co-culture systems with ligand-expressing cells to activate endogenous Notch signaling

What are common challenges when working with N4BP1 antibodies and how can they be addressed?

Researchers may encounter several challenges when using N4BP1 antibodies:

• Multiple Bands in Western Blots:

  • N4BP1 undergoes various post-translational modifications including ubiquitination and SUMOylation

  • It can also be cleaved by caspase-8 during inflammatory responses

  • Solution: Use positive controls with known N4BP1 expression; include protease inhibitors in lysis buffers; consider specific treatments (e.g., phosphatase) to identify the nature of modifications

• Subcellular Localization Variability:

  • N4BP1 primarily localizes to nucleoli but is also found in a subset of PML nuclear bodies

  • Its localization may change under different cellular conditions

  • Solution: Use co-staining with nucleolar markers (fibrillarin) and PML to confirm authentic localization patterns

• Low Signal in Immunoprecipitation:

  • N4BP1 interactions may be transient or condition-dependent

  • Solution: Consider crosslinking approaches; optimize buffer conditions; increase antibody concentration; use tagged versions of N4BP1 for difficult interactions

How can contradictory results regarding N4BP1 function be reconciled?

N4BP1 exhibits context-dependent functions that may appear contradictory:

• Cell Type Specificity:

  • N4BP1's effects may differ between cell types (e.g., immune cells versus neuronal cells)

  • Solution: Always specify the cell type used and avoid generalizing findings across systems

• Stimulus-Dependent Effects:

  • N4BP1 shows different behaviors depending on the triggering stimulus (e.g., TLR activation patterns)

  • Solution: Carefully control experimental conditions and stimulus parameters

• Regulatory Mechanisms:

  • N4BP1 itself is regulated by cleavage, ubiquitination, and other modifications

  • Solution: Monitor N4BP1 status (intact vs. cleaved) when interpreting functional outcomes

• Domain-Specific Functions:

  • Different domains of N4BP1 mediate distinct functions (RNA binding, ubiquitin interaction)

  • Solution: Use domain-specific mutants to dissect which activity is relevant to the observed phenotype

What controls are essential when studying N4BP1 in inflammatory signaling?

When investigating N4BP1's role in inflammatory pathways:

• Pathway-Specific Controls:

  • Include both TRIF-dependent (TLR3, TLR4) and TRIF-independent (TLR2, TLR7, TLR9) stimulation

  • N4BP1 specifically enhances TRIF-independent NF-κB activation

• Temporal Controls:

  • Monitor signaling at multiple time points, as N4BP1 regulates the duration of proinflammatory signaling

  • Include both early (minutes) and late (hours) timepoints after stimulation

• Genetic Controls:

  • Use N4BP1-deficient cells alongside wild-type controls

  • Complement with rescue experiments using wild-type and mutant forms of N4BP1

  • Consider caspase-8 inhibitors or knockdown to prevent N4BP1 cleavage

• Readout Controls:

  • Measure multiple inflammatory outputs (NF-κB activation, cytokine production)

  • Include known target genes like IL-6 and CXCL1

  • Validate key findings using both in vitro and in vivo models when possible

How can antibodies help uncover N4BP1's role in viral restriction?

N4BP1 has been identified as a restriction factor against some viruses, including HIV-1 . To investigate this function:

• Viral Replication Assays:

  • Compare viral titers or reporter gene expression in cells with normal or depleted N4BP1 levels

  • Use immunofluorescence to detect colocalization of N4BP1 with viral components

  • Examine N4BP1 recruitment to viral RNA using RIP or CLIP techniques

• RNA Degradation Analysis:

  • Assess whether N4BP1's ribonuclease activity targets viral RNA specifically

  • Compare N4BP1 wild-type with the D623N mutant that lacks endoribonuclease activity

  • Measure half-lives of viral transcripts in the presence or absence of N4BP1

• Mechanistic Studies:

  • Investigate whether viral infection induces changes in N4BP1 expression, localization, or post-translational modifications

  • Determine if viruses have evolved countermeasures against N4BP1 restriction

What new techniques are emerging to study N4BP1 dimerization and its LUBIN domain?

Recent research has identified N4BP1 dimerization as critical for its function as a linear ubiquitin reader . To investigate this:

• Structural Analysis:

  • Use cross-linking mass spectrometry to identify dimerization interfaces

  • Apply proximity labeling techniques (BioID, APEX) to map spatial relationships in the dimer

  • Consider cryo-EM for structural determination of the N4BP1 dimer bound to linear ubiquitin chains

• Functional Assays:

  • Generate dimerization-deficient mutants to assess the impact on linear ubiquitin binding

  • Develop FRET-based sensors to monitor dimerization dynamics in living cells

  • Use size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to characterize dimerization status

• Disease Relevance:

  • Investigate whether disease-associated mutations affect N4BP1 dimerization or LUBIN function

  • Examine whether targeting the dimerization interface could have therapeutic potential in inflammatory conditions