NRG4 Antibody

What is NRG4 and what tissues express it most abundantly?

NRG4 is a member of the neuregulin family that functions as a ligand for the ErbB4 receptor tyrosine kinase. It plays crucial roles in metabolic regulation and energy homeostasis. Expression analysis reveals a highly tissue-specific pattern:

• Highly enriched in brown adipose tissue (BAT)

• Present at lower levels in white adipose tissue (WAT)

• Relatively low expression in skeletal muscle, liver, brain, and heart

• Expression in BAT is elevated by acute cold exposure

• Expression in both BAT and inguinal WAT is augmented by cold acclimation

NRG4 undergoes proteolytic cleavage near the transmembrane domain to release biologically active extracellular fragments that act through autocrine, paracrine and endocrine mechanisms . This tissue-specific expression pattern makes NRG4 a valuable target for metabolic research.

What are the molecular characteristics of the NRG4 protein?

NRG4 has several distinctive molecular features that researchers should consider when designing experiments:

The released extracellular fragments contain a highly conserved N-terminal EGFL peptide that mediates receptor binding . The protein undergoes proteolytic cleavage to release its biologically active form, making it important to consider whether your research requires detection of the membrane-bound or secreted form.

How do I select the appropriate NRG4 antibody for my research?

When selecting an NRG4 antibody, consider these critical factors:

• Research application: Determine if you need antibodies validated for Western blot, IHC, ELISA, or other applications

• Species reactivity: Verify the antibody reacts with your species of interest (human, mouse, rat)

• Target epitope: Consider whether you need to detect the full-length protein or the cleaved/secreted fragment

• Clonality: Polyclonal antibodies may offer broader epitope recognition, while monoclonals provide higher specificity

• Validation data: Review provided validation images (Western blots, IHC staining)

• Control samples: Plan to include positive controls like brown adipose tissue or lung tissue

Antibody selection significantly impacts experimental outcomes, particularly for NRG4 which exists in both membrane-bound and secreted forms. Request validation data specific to your application before purchasing.

How does NRG4 signaling differ from other neuregulins in receptor specificity and downstream effects?

NRG4 demonstrates unique receptor specificity compared to other neuregulin family members:

• NRG4 specifically activates ErbB4 receptors, unlike NRG1 which activates both ErbB3 and ErbB4

• This specificity allows selective activation of ErbB4-ErbB4 homodimers rather than ErbB4-ErbB2 heterodimers

• In breast cancer research, NRG1 shows both pro- and anti-proliferative effects, whereas NRG4 consistently shows anti-proliferative effects when combined with anti-HER2 agents

• NRG4 binding to ErbB4 elicits strong tyrosine phosphorylation of the receptor

This receptor specificity makes NRG4 a valuable tool for distinguishing between ErbB3 and ErbB4 signaling pathways. The differential effects of NRG4 versus NRG1 in cancer models suggest distinct downstream signaling mechanisms that warrant further investigation for therapeutic applications.

What is the role of NRG4 in breast cancer progression and treatment?

Research indicates NRG4 has significant potential in breast cancer treatment strategies:

• Higher ERBB4 levels (the receptor for NRG4) correlate with longer relapse-free survival in HER2-enriched and luminal A breast cancer subtypes

• NRG4 treatment robustly potentiates the anti-proliferative action of anti-HER2 agents like trastuzumab and pertuzumab in HER2+ breast cancer cells

• In SKBR3 cells, combination of NRG4 with pertuzumab produced a dramatic 98% reduction in cell number

• Unlike NRG1, which can reduce the efficacy of anti-HER2 agents, NRG4 consistently enhances their effectiveness

These findings suggest a paradigm shift in cancer treatment approaches, proposing the administration of ERBB4 ligands like NRG4 to improve the efficacy of anti-ERBB2 agents . This represents a novel strategy that activates ERBB4-ERBB4 homodimers to enhance therapeutic outcomes in specific breast cancer subtypes.

How do experimental conditions affect NRG4 protein detection in different tissue types?

Several experimental factors significantly impact NRG4 detection:

• Cold exposure effects: NRG4 expression in BAT increases with acute cold exposure, while expression in both BAT and inguinal WAT increases with cold acclimation

• Cell differentiation stage: NRG4 expression is markedly induced during adipocyte differentiation

• Adrenergic stimulation: NRG4 is induced by norepinephrine in differentiated brown adipocytes but not in 3T3-L1 white adipocytes

• Tissue processing: Due to its low molecular weight and potential for proteolytic processing, sample preparation methods are critical

• Detection of secreted forms: Analysis of conditioned media may be necessary to capture the secreted form of NRG4

These variables must be carefully controlled in experimental design. For studies involving adipose tissue, documenting temperature conditions and considering the effects of adrenergic stimulation are particularly important for generating reproducible results.

What are the optimal protocols for detecting NRG4 expression by Western blotting?

For successful Western blot detection of NRG4:

• Sample preparation:

  • Use lysis buffers containing appropriate detergents (NP-40, Triton X-100) for membrane protein extraction

  • Include protease inhibitors to prevent degradation

  • For signaling studies, add phosphatase inhibitors (e.g., Na₃VO₄ at 1mM)

• Gel electrophoresis and transfer:

  • Use 15-20% gels for optimal resolution of this low molecular weight protein

  • Transfer to nitrocellulose membrane (0.45 μm pore size)

• Antibody incubation:

  • Block membranes with 5% BSA in TBS-T

  • Use NRG4 antibodies at dilutions of 1:500-1:1000

  • Incubate overnight at 4°C for primary antibody

  • Use HRP-conjugated secondary antibodies matched to the primary antibody host

• Detection:

  • Use enhanced chemiluminescence detection systems

  • Expect bands at approximately 12.7-13 kDa

Including positive controls such as brown adipose tissue or lung tissue lysates is essential for validating specific binding . Western blotting is particularly useful for distinguishing between the membrane-bound and cleaved forms of NRG4.

How should researchers troubleshoot specificity issues with NRG4 antibodies?

When encountering specificity issues with NRG4 antibodies, implement these troubleshooting approaches:

• Validation experiments:

  • Test the antibody on known positive controls (brown adipose tissue, differentiated brown adipocytes)

  • Include negative controls with low NRG4 expression (confirmed by other methods)

  • Perform peptide competition assays with the immunizing peptide

• Cross-validation strategies:

  • Compare results from multiple antibodies targeting different epitopes of NRG4

  • Verify that detected protein size matches the expected molecular weight (12.7-13 kDa)

  • Confirm expression using orthogonal methods (qPCR, mass spectrometry)

• Optimization approaches:

  • Test a range of antibody dilutions beyond the recommended range

  • Modify blocking conditions to reduce background (test both BSA and non-fat milk)

  • Increase washing stringency to reduce non-specific binding

• Technical controls:

  • Use recombinant NRG4 protein as a positive control

  • Compare results in wild-type versus NRG4 knockout or knockdown models

Addressing specificity issues requires systematic investigation of both experimental conditions and antibody characteristics. Document all optimization steps thoroughly to ensure reproducibility.

What are the key considerations for immunohistochemical detection of NRG4?

For effective immunohistochemical analysis of NRG4:

• Tissue preparation:

  • Proper fixation is critical; overfixation may mask epitopes

  • For paraffin-embedded tissues, optimize antigen retrieval methods

  • Consider tissue-specific expression patterns when selecting positive controls

• Antibody selection and dilution:

  • Use antibodies specifically validated for IHC applications

  • Start with recommended dilutions (typically 1:50-1:200 for IHC-P)

  • Consider the domain specificity of the antibody (extracellular vs. intracellular)

• Controls and validation:

  • Include known positive tissues (brown adipose tissue, prostate cancer samples)

  • Use isotype controls to assess non-specific binding

  • Consider serial dilutions of primary antibody to determine optimal concentration

• Signal detection and interpretation:

  • Document subcellular localization patterns (membrane, cytoplasmic)

  • Be aware that NRG4 may exhibit both membrane and secreted forms

  • Use appropriate image acquisition settings to capture the true signal range

The expression of NRG4 has been studied in prostate cancer using IHC staining, providing valuable precedent for cancer-related applications . Consider co-staining with cell type-specific markers to better characterize the cells expressing NRG4 in heterogeneous tissues.

How can NRG4 be utilized as a therapeutic target in metabolic disorders?

NRG4's role in metabolic regulation offers several therapeutic directions:

• NRG4 protects against diet-induced insulin resistance and hepatic steatosis through attenuating hepatic lipogenic signaling

• As a brown fat-enriched secreted factor, NRG4 may serve as a potential therapeutic for metabolic disorders like obesity and diabetes

• NRG4 is involved in regulating lipid metabolism and glucose homeostasis

• The protein's restricted expression pattern makes it a potentially specific target with fewer off-target effects

Research approaches might include:

• Development of recombinant NRG4 or NRG4-mimetic compounds for therapeutic administration

• Gene therapy approaches targeting increased NRG4 expression in brown adipose tissue

• Drug discovery targeting the NRG4-ErbB4 signaling pathway

• Combination approaches with existing metabolic disorder treatments

Understanding the complete signaling mechanisms downstream of NRG4 activation remains a critical area for future research to fully exploit its therapeutic potential.

What experimental approaches can be used to study NRG4-ErbB4 signaling dynamics?

To investigate NRG4-ErbB4 signaling dynamics, researchers can employ these approaches:

• Receptor activation studies:

  • Monitor ErbB4 phosphorylation after NRG4 treatment using phospho-specific antibodies

  • Compare activation kinetics between NRG4 and other neuregulin family members

  • Use conditioned media from NRG4-expressing cells to activate ErbB4 in target cells

• Pathway analysis:

  • Investigate downstream signaling using phospho-specific antibodies against key pathway components

  • Employ selective pathway inhibitors to delineate specific signaling branches

  • Conduct temporal analyses to map signaling dynamics over time

• Advanced techniques:

  • Use CRISPR/Cas9-mediated gene editing to create receptor variants

  • Employ proximity ligation assays to study receptor dimerization patterns

  • Utilize live-cell imaging with fluorescently tagged proteins to monitor receptor trafficking

• Functional readouts:

  • Assess cell proliferation, migration, or metabolic changes in response to NRG4

  • Compare the effects of NRG4 versus NRG1 on cell behavior and signaling

  • Measure the influence of NRG4 on the efficacy of other therapeutic agents

These approaches can help elucidate the unique aspects of NRG4-ErbB4 signaling and its therapeutic implications in both metabolic disorders and cancer.

How do you design experiments to differentiate between the effects of membrane-bound versus secreted NRG4?

Designing experiments to distinguish between membrane-bound and secreted NRG4 requires careful consideration:

• Experimental design strategies:

  • Use domain-specific antibodies that distinguish between full-length and cleaved forms

  • Create mutant constructs with altered cleavage sites to prevent proteolytic processing

  • Employ SEAP-NRG4 fusion proteins to facilitate detection of cleaved fragments in media

  • Compare effects of recombinant soluble NRG4 versus cell-expressed full-length NRG4

• Cell culture approaches:

  • Collect and concentrate conditioned media to analyze secreted forms

  • Create stable cell lines expressing wild-type or cleavage-resistant NRG4

  • Use transwell co-culture systems to distinguish paracrine from autocrine effects

• Analytical methods:

  • Fractionate samples to separate membrane fractions from soluble proteins

  • Employ size exclusion chromatography to distinguish different forms

  • Use domain-specific antibodies in Western blot to identify specific fragments

• Functional comparisons:

  • Compare receptor activation efficiency between membrane-bound and soluble forms

  • Assess differences in downstream signaling pathways activated by each form

  • Evaluate cell-type specific responses to different NRG4 forms

Understanding the biological differences between membrane-bound and secreted NRG4 is critical for developing targeted therapeutic approaches and interpreting experimental results accurately.

What is the potential for using NRG4 in combination therapies for breast cancer treatment?

Recent research reveals promising directions for NRG4 in combination cancer therapies:

• NRG4 robustly potentiates the anti-proliferative action of trastuzumab and pertuzumab in HER2+ breast cancer cells

• Unlike NRG1, which can reduce anti-HER2 agent efficacy, NRG4 consistently enhances their effectiveness

• In BT474 cells, NRG4 boosted trastuzumab efficacy, reducing cell number and increasing cell doubling time

• In SKBR3 cells, the combination of NRG4 and pertuzumab showed dramatic synergy with 98% reduction in cell number after 3 days

These findings suggest a novel therapeutic approach combining NRG4 with established anti-ERBB2 neutralizing antibodies. This represents a paradigm shift in cancer treatment strategies, where a growth factor (NRG4) is administered to improve anti-cancer efficacy rather than being targeted for inhibition .

Future research should focus on optimizing dosing regimens, delivery methods, and identifying biomarkers that predict response to combination therapy.

How can researchers quantify circulating NRG4 in plasma or serum samples?

Quantifying circulating NRG4 presents significant challenges:

• Despite significant efforts, highly sensitive and specific immunological assays for NRG4 detection in plasma remain challenging to develop

• The relatively low molecular weight and potential post-translational modifications complicate detection

• Cleaved fragments may have different half-lives in circulation compared to the full-length protein

Recommended approaches include:

• Method development considerations:

  • Develop sandwich ELISA assays using antibodies targeting different epitopes

  • Consider enrichment techniques like immunoprecipitation before detection

  • Evaluate mass spectrometry-based approaches for unambiguous identification

• Sample handling:

  • Standardize collection procedures to minimize proteolytic degradation

  • Consider the timing of sample collection relative to metabolic state

  • Use appropriate protease inhibitors during sample processing

• Validation approaches:

  • Compare levels in conditions known to affect NRG4 expression (e.g., metabolic disorders)

  • Include spike-in controls of recombinant NRG4 to assess recovery

  • Develop reference ranges using samples from well-characterized cohorts

The development of reliable assays for circulating NRG4 would significantly advance both basic research and clinical applications, particularly for metabolic disorders and cancer research.