Axin1 Antibody

What is the biological significance of Axin1 in cellular signaling pathways?

Axin1 functions as a crucial scaffold protein in multiple signaling cascades. It is primarily recognized as a component of the beta-catenin destruction complex that regulates CTNNB1 levels through phosphorylation and ubiquitination, thus modulating Wnt-signaling pathways . Additionally, Axin1 controls dorsoventral patterning through two opposing mechanisms: it down-regulates CTNNB1 to inhibit the Wnt signaling pathway while simultaneously activating a Wnt-independent JNK signaling pathway . Beyond its role in Wnt signaling, Axin1 enhances TGF-beta signaling by recruiting the RNF111 E3 ubiquitin ligase and promoting the degradation of inhibitory SMAD7 . Axin1 also partners with HIPK2 and TP53 to form a complex that regulates cell growth, apoptosis, and development, particularly in response to cellular stresses like ultraviolet irradiation .

How do Axin1 and Axin2 differ functionally in experimental models?

While both Axin1 and Axin2 are scaffold proteins that can negatively regulate Wnt signaling, they exhibit distinct functional properties in various experimental contexts. Research has demonstrated that overexpression of Axin1, but not Axin2, inhibits viral nucleoprotein (NP), hemagglutinin (HA), and matrix protein (M1) mRNA levels during influenza virus infection . This functional differentiation extends to other viral models, where Axin1 overexpression significantly reduces respiratory syncytial virus (RSV) viral G and M protein levels at 36 and 48 hours post-infection, while Axin2 overexpression does not produce comparable effects . Furthermore, Axin1 uniquely boosts type I interferon responses during viral infection, significantly increasing IFNβ1 mRNA expression and augmenting the expression of interferon-stimulated genes like OAS1 . These differences highlight the importance of selecting the appropriate Axin protein for specific experimental objectives.

What molecular weight should be expected when detecting Axin1 in Western blot applications?

When performing Western blot analysis of Axin1, researchers should expect some variability in the observed molecular weight. The calculated molecular weight based on amino acid sequence is approximately 92-95 kDa (826aa/92 kDa or 862aa/95 kDa), but the observed molecular weight typically ranges between 100-110 kDa . This discrepancy is attributable to post-translational modifications that Axin1 undergoes, particularly poly(ADP-ribosyl)ation by tankyrases TNKS and TNKS2 . When conducting Western blots, it is advisable to include positive controls from validated cell lines such as HeLa, HT-1080, or HEK-293T cells, which have been confirmed to express detectable levels of Axin1 . The apparent molecular weight may vary slightly depending on the specific tissues or cell types being examined due to differential post-translational modifications.

What are the optimal conditions for immunohistochemistry applications using Axin1 antibodies?

For successful immunohistochemistry (IHC) applications with Axin1 antibodies, optimal antigen retrieval and antibody dilution are critical factors. Evidence-based protocols recommend antigen retrieval with TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative option . The recommended antibody dilution range for IHC applications is 1:50-1:500, but this should be optimized for each specific experimental system and tissue type . Human colon tissue has been validated as a positive control for IHC applications with Axin1 antibodies .

When conducting IHC with Axin1 antibodies, researchers should implement the following systematic approach:

It is advisable to include both positive and negative controls in each experimental run to ensure specificity and validate staining patterns.

How should researchers optimize Western blot protocols for detecting Axin1?

For optimal Western blot detection of Axin1, several methodological considerations must be addressed. The recommended antibody dilution range is 1:1000-1:6000, though this should be titrated for each specific experimental system . When preparing samples, complete protease inhibitor cocktails should be included to prevent degradation of Axin1, which can be susceptible to proteolysis. The selection of an appropriate protein transfer method is also crucial—semi-dry transfer systems may be less effective than wet transfer for this relatively large protein (100-110 kDa) .

A comprehensive protocol optimization strategy should include:

• Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors, and potential tankyrase inhibitors (e.g., XAV939) to stabilize Axin1 levels

• Gel selection: Use 8-10% polyacrylamide gels for optimal resolution of Axin1

• Transfer conditions: Wet transfer at 30V overnight at 4°C is recommended for complete transfer

• Blocking: 5% non-fat milk in TBST for 1-2 hours at room temperature

• Primary antibody incubation: Dilute antibody in 5% BSA in TBST and incubate overnight at 4°C

• Visualization: Use enhanced chemiluminescence systems with sufficient sensitivity for detecting potentially low endogenous levels of Axin1

HeLa, HT-1080, and HEK-293T cell lysates serve as reliable positive controls for Axin1 Western blot applications .

What experimental controls are essential when using Axin1 antibodies for validating knockout or knockdown studies?

When conducting Axin1 knockout (KO) or knockdown (KD) validation studies, implementing appropriate controls is fundamental to ensuring experimental rigor. A comprehensive validation approach requires multiple complementary controls:

• Positive expression controls: Include wild-type cell lysates or tissue samples known to express Axin1 (e.g., HeLa, HT-1080, or HEK-293T cells)

• Negative controls: Incorporate verified Axin1 KO cell lines when available

• Knockdown specificity controls: When using siRNA, include scrambled siRNA controls and monitor Axin2 levels to confirm specificity of Axin1 knockdown, as research has shown that knockdown of Axin1 typically does not affect Axin2 levels

• Functional validation: Assess downstream effects on beta-catenin levels or JNK/c-Jun phosphorylation status to confirm functional consequences of Axin1 depletion

• Rescue experiments: Reintroduce wild-type Axin1 to KO/KD systems to demonstrate reversal of phenotypes

• Multiple antibody validation: Use at least two different Axin1 antibodies targeting distinct epitopes to confirm specificity of detection

This multilayered approach to experimental controls ensures robust validation of Axin1 modulation and minimizes the risk of misinterpreting experimental outcomes due to off-target effects or antibody cross-reactivity.

How can Axin1 antibodies be utilized to investigate Wnt signaling pathway dysregulation in cancer models?

Axin1 antibodies provide powerful tools for investigating Wnt signaling dysregulation in cancer models due to Axin1's function as a tumor suppressor and critical component of the beta-catenin destruction complex . A sophisticated experimental approach involves:

• Comparative expression analysis: Utilize Axin1 antibodies in Western blot and IHC applications to quantitatively compare Axin1 expression levels between normal and malignant tissues. Recommended antibody dilutions are 1:1000-1:6000 for Western blot and 1:50-1:500 for IHC applications .

• Co-immunoprecipitation studies: Apply Axin1 antibodies in co-IP experiments to assess changes in the composition of the beta-catenin destruction complex (including GSK3B and APC) in cancer cells versus normal controls.

• Phosphorylation status assessment: Combine Axin1 antibodies with phospho-specific antibodies to evaluate changes in the phosphorylation status of Axin1 and its binding partners.

• Proteasomal degradation analysis: Use Axin1 antibodies in conjunction with proteasome inhibitors (e.g., MG132) to investigate alterations in Axin1 stability and turnover in cancer cells.

• Subcellular localization studies: Employ Axin1 antibodies in immunofluorescence microscopy to track changes in subcellular distribution of Axin1 during malignant transformation.

• Interactome mapping: Utilize Axin1 antibodies for proximity ligation assays or mass spectrometry-based interactome studies to identify cancer-specific alterations in Axin1's protein-protein interaction network.

This multifaceted approach enables researchers to comprehensively characterize Wnt signaling dysregulation mechanisms in cancer development and progression.

What methodologies can be employed to study the role of Axin1 in antiviral immune responses?

Recent research has uncovered a novel role for Axin1 in antiviral immunity, particularly in response to influenza virus infection . To investigate this function, researchers can implement the following methodological approaches:

• Viral challenge models with Axin1 modulation: Overexpress Axin1 using expression vectors or stabilize endogenous Axin1 using tankyrase inhibitors like XAV939 prior to viral infection. Research has demonstrated that both approaches significantly reduce influenza virus replication in lung epithelial cells, with similar effects observed for respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) .

• Interferon response assessment: Measure mRNA expression of IFNβ1 and interferon-stimulated genes (e.g., OAS1) using qRT-PCR in Axin1-overexpressing versus control cells following viral infection. Data indicates that Axin1 markedly increases IFNβ1 expression in virus-infected cells but not in uninfected cells .

• STAT1 phosphorylation analysis: Utilize phospho-specific antibodies to assess STAT1 phosphorylation levels in Western blot applications, as Axin1 has been shown to increase STAT1 phosphorylation upon viral infection .

• Signaling pathway inhibition studies: Employ pathway-specific inhibitors (e.g., ruxolitinib for JAK1/2 or fludarabine for STAT1) to determine whether the Axin1-mediated antiviral effect depends on interferon signaling. Research has demonstrated that these inhibitors reverse Axin1-mediated antiviral effects .

• In vivo infection models: Administer XAV939 to stabilize Axin1 in animal models prior to viral challenge to assess protection against lethal influenza virus infection, as XAV939 has been shown to protect mice from influenza virus infection .

This comprehensive experimental strategy enables detailed characterization of Axin1's role in antiviral immunity and interferon responses.

How can researchers investigate the interplay between Axin1 and the JNK/c-Jun signaling pathway?

Axin1 has been identified as a crucial regulator of the JNK/c-Jun signaling pathway, which contributes to its role in both developmental processes and antiviral responses . To elucidate this interplay, researchers can implement the following methodological approach:

• Co-immunoprecipitation studies: Utilize Axin1 antibodies to isolate Axin1-containing protein complexes, followed by Western blot analysis to detect JNK and c-Jun association. This approach can identify direct physical interactions between Axin1 and components of the JNK signaling cascade.

• Phosphorylation analysis: Assess JNK and c-Jun phosphorylation levels in response to Axin1 overexpression or knockdown using phospho-specific antibodies. Research has demonstrated that Axin1 activates JNK/c-Jun signaling, particularly during viral infection .

• Functional domain mapping: Express truncated Axin1 constructs lacking specific domains to determine which regions are essential for JNK/c-Jun pathway activation. This approach can identify the specific structural elements of Axin1 required for JNK pathway regulation.

• Pathway inhibitor studies: Utilize JNK-specific inhibitors (e.g., SP600125) to determine whether the observed effects of Axin1 overexpression are dependent on JNK signaling. This approach can help establish causality in the relationship between Axin1 and JNK pathway activation.

• Comparative analysis with Axin2: Since research has shown functional differences between Axin1 and Axin2 in various contexts , comparing their effects on JNK/c-Jun signaling can provide insights into isoform-specific functions.

• Transcriptional reporter assays: Employ AP-1 luciferase reporter constructs to quantitatively measure c-Jun transcriptional activity in response to Axin1 modulation, providing a functional readout of pathway activation.

This comprehensive experimental approach enables detailed characterization of the molecular mechanisms underlying Axin1's regulation of the JNK/c-Jun signaling pathway.

How should researchers address inconsistent Axin1 detection in Western blot applications?

Inconsistent detection of Axin1 in Western blot applications is a common challenge that can stem from multiple factors. A systematic troubleshooting approach includes:

• Sample preparation optimization: Axin1 is subject to degradation through normal cellular turnover mechanisms, including poly(ADP-ribosyl)ation by tankyrases TNKS and TNKS2 . Include tankyrase inhibitors like XAV939 in lysis buffers to stabilize Axin1 levels. Research has shown that XAV939 markedly increases Axin1 protein levels in both the presence and absence of influenza virus infection .

• Antibody selection and validation: Antibody validation is critical for reliable Axin1 detection. Test multiple antibodies targeting different epitopes of the protein. The observed molecular weight for Axin1 typically ranges between 100-110 kDa despite a calculated weight of 92-95 kDa due to post-translational modifications .

• Positive control inclusion: Always include validated positive controls such as HeLa, HT-1080, or HEK-293T cell lysates, which have been confirmed to express detectable levels of Axin1 .

• Transfer conditions optimization: For large proteins like Axin1 (100-110 kDa), extend transfer times or decrease voltage to ensure complete transfer to the membrane. Wet transfer systems often yield better results than semi-dry systems for proteins of this size.

• Detection system sensitivity: Axin1 may be expressed at relatively low levels in some tissues or cell types. Use high-sensitivity detection reagents and consider longer exposure times when necessary.

• Quantification standardization: When comparing Axin1 levels between samples, normalize to loading controls and include internal reference standards to account for blot-to-blot variation.

This comprehensive troubleshooting approach addresses the most common sources of inconsistency in Axin1 detection.

What approaches can resolve discrepancies between Axin1 protein levels and functional outcomes in experimental models?

Researchers may encounter situations where observed Axin1 protein levels do not directly correlate with expected functional outcomes. Resolving these discrepancies requires a multifaceted approach:

• Post-translational modification analysis: Axin1 function is heavily regulated by post-translational modifications. Investigate phosphorylation status using phospho-specific antibodies or mass spectrometry, as phosphorylation can alter Axin1's functional properties without changing total protein levels.

• Subcellular localization assessment: The functional impact of Axin1 depends on its subcellular localization. Utilize cellular fractionation followed by Western blot or immunofluorescence microscopy to determine whether Axin1 redistribution, rather than total level changes, explains functional discrepancies.

• Complex formation evaluation: Axin1 functions as a scaffold protein in multiprotein complexes. Investigate changes in complex composition using co-immunoprecipitation followed by Western blot or mass spectrometry to determine whether alterations in protein-protein interactions explain functional differences despite similar total Axin1 levels.

• Pathway crosstalk consideration: Axin1 participates in multiple signaling pathways, including Wnt, JNK/c-Jun, and TGF-beta signaling . Changes in one pathway may compensate for or override effects in another pathway. Comprehensively assess all relevant downstream pathways to identify compensatory mechanisms.

• Isoform-specific analysis: Different Axin1 isoforms may have distinct functional properties. Use isoform-specific antibodies or RT-PCR to determine whether changes in isoform distribution explain functional discrepancies despite similar total Axin1 protein levels.

This systematic approach enables researchers to reconcile apparent discrepancies between Axin1 protein levels and functional outcomes in their experimental models.

How can researchers differentiate between direct and indirect effects of Axin1 modulation in cellular systems?

Distinguishing direct effects of Axin1 modulation from secondary consequences is methodologically challenging but critical for accurate data interpretation. A comprehensive strategy includes:

• Temporal analysis: Implement time-course experiments to establish the sequence of events following Axin1 modulation. Direct effects typically manifest earlier than indirect downstream consequences. For example, research has shown that Axin1 overexpression leads to increased IFNβ1 expression specifically in virus-infected cells but not in uninfected cells, suggesting a context-dependent direct effect .

• Pathway inhibitor studies: Utilize specific inhibitors of known Axin1-associated pathways (e.g., Wnt, JNK, TGF-beta) to determine whether the observed effects depend on these signaling cascades. In antiviral studies, JAK1/2 inhibitor ruxolitinib or STAT1 inhibitor fludarabine reversed the Axin1-mediated antiviral effects, demonstrating that these effects depend on interferon signaling .

• Structure-function analysis: Express mutant Axin1 proteins with alterations in specific functional domains to identify which regions are necessary for particular cellular responses. This approach can establish direct mechanistic links between Axin1 and observed phenotypes.

• Proximal interaction mapping: Implement proximity ligation assays or BioID approaches to identify proteins that directly interact with Axin1 following specific stimuli or treatments, providing insights into the initial events in Axin1-mediated signaling.

• Acute induction systems: Utilize inducible expression systems (e.g., Tet-On) or rapid protein degradation approaches (e.g., auxin-inducible degron system) to achieve temporal control over Axin1 levels, allowing better discrimination between immediate and delayed effects.

• In vitro reconstitution: For suspected direct biochemical effects, attempt to reconstitute the relevant reactions using purified components in cell-free systems to eliminate cellular complexity and establish direct causality.

This methodological framework enables researchers to differentiate between direct and indirect consequences of Axin1 modulation, allowing more precise mechanistic interpretations of experimental results.

What are the potential applications of Axin1 stabilizers like XAV939 in antiviral therapeutic development?

The discovery that Axin1 stabilization confers antiviral protection opens promising avenues for therapeutic development. The tankyrase inhibitor XAV939, which stabilizes Axin1 by preventing its poly(ADP-ribosyl)ation and subsequent degradation, has demonstrated significant antiviral activity against multiple viruses including influenza, respiratory syncytial virus (RSV), and vesicular stomatitis virus (VSV) . Researchers investigating the therapeutic potential of Axin1 stabilization should consider the following methodological approaches:

• Broad-spectrum activity assessment: Evaluate XAV939 and other Axin1 stabilizers against diverse virus families to determine the scope of antiviral activity. Research has already demonstrated efficacy against influenza virus and RSV , suggesting potential broad-spectrum applications.

• In vivo efficacy studies: Conduct animal model studies with various administration routes and treatment timings. Research has shown that XAV939 protects mice from lethal influenza virus challenge, providing proof-of-concept for in vivo efficacy .

• Mechanism of action characterization: Investigate whether the antiviral effects rely exclusively on interferon response enhancement or involve additional mechanisms. JAK1/2 and STAT1 inhibitors reverse XAV939-mediated antiviral effects, confirming the central role of interferon signaling in its mechanism of action .

• Resistance development assessment: Evaluate the potential for viruses to develop resistance to Axin1 stabilizer-based therapies through serial passage experiments. Host-targeted approaches like Axin1 stabilization may present higher barriers to resistance than direct-acting antivirals.

• Combination therapy exploration: Test Axin1 stabilizers in combination with established antivirals to identify potential synergistic effects that could enhance therapeutic outcomes while minimizing the risk of resistance development.

• Safety profile characterization: Comprehensively assess potential off-target effects and safety implications of prolonged Axin1 stabilization, considering its roles in multiple cellular signaling pathways including Wnt, JNK/c-Jun, and TGF-beta.

This systematic research approach will advance understanding of Axin1 stabilizers as potential broad-spectrum antiviral therapeutics.

How can researchers explore the interplay between Axin1 and other scaffold proteins in integrated cellular signaling networks?

Axin1 functions within complex cellular signaling networks, interacting with multiple pathways including Wnt, JNK/c-Jun, TGF-beta, and interferon signaling . Investigating its interplay with other scaffold proteins requires sophisticated experimental approaches:

• Proximity-based interactome mapping: Implement BioID or APEX2 proximity labeling techniques with Axin1 as the bait protein to identify proteins that physically associate with Axin1 in living cells under various conditions. This approach can reveal context-dependent interactions with other scaffold proteins.

• Competitive binding studies: Investigate whether overexpression of other scaffold proteins affects Axin1's ability to form functional complexes or activate downstream pathways. This approach can identify instances of competition for shared binding partners.

• Pathway crosstalk quantification: Simultaneously monitor multiple signaling pathways (e.g., Wnt, JNK, TGF-beta, interferon) following Axin1 modulation to construct integrated network models. Research has shown that Axin1 simultaneously regulates multiple pathways, including enhancing TGF-beta signaling and activating JNK/c-Jun signaling .

• Combinatorial knockdown/knockout experiments: Implement simultaneous modulation of Axin1 and other scaffold proteins to identify synthetic interactions that suggest functional relationships between distinct signaling modules.

• Dynamic interaction mapping: Utilize fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) approaches to monitor real-time changes in Axin1's interactions with other scaffold proteins following specific stimuli.

• Computational network modeling: Integrate experimental data into computational models that can predict emergent properties of signaling networks containing multiple scaffold proteins, including Axin1.

This comprehensive experimental strategy enables researchers to elucidate how Axin1 functions within the broader context of cellular signaling networks and how its interactions with other scaffold proteins contribute to integrated cellular responses.