SHOX2 Antibody

What is SHOX2 and what are its primary biological functions?

SHOX2 (Short stature homeobox 2) is a transcription factor that plays essential roles in multiple biological processes. It functions as a growth regulator and participates in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation . SHOX2 also interfaces with T-box proteins like TBX5, contributing to the specification and function of heart tissues, ensuring proper formation of heart structures, skeletal growth, and limb patterning .

Beyond these roles, SHOX2 is a molecular determinant of depot-specific adipocyte function, with higher expression in subcutaneous compared to visceral fat in both rodents and humans . This differential expression influences adipocyte metabolism and lipolysis rates through regulation of β3 adrenergic receptor (Adrb3) expression . Additionally, SHOX2 is critical for cardiac pacemaker function and atrioventricular conduction, making it important in cardiac electrophysiology research .

What types of SHOX2 antibodies are available for research?

Researchers have access to several types of SHOX2 antibodies with varying characteristics:

Monoclonal Antibodies:

• Mouse monoclonal antibodies such as SHOX2 antibody [1D1] (ab55740), which is suitable for Western blot and IHC-Fr applications and reacts with human and rat samples .

• Mouse IgG 2a κ antibodies like JK-6E (sc-81955) that target epitopes within amino acids 117-205 of human SHOX2 and are applicable for WB, IP, and ELISA techniques .

Polyclonal Antibodies:

• Rabbit polyclonal antibodies like 16366-1-AP that target SHOX2 in WB, IHC, and ELISA applications, showing reactivity with human, mouse, and rat samples .

These antibodies differ in their specificity, target epitopes, and optimal applications, providing researchers with options based on their experimental needs.

What are the standard applications for SHOX2 antibodies?

SHOX2 antibodies can be utilized in multiple research applications:

When performing Western blot analysis, SHOX2 typically appears as a band at approximately 34-40 kDa . For IHC applications, antigen retrieval methods may influence results, with TE buffer pH 9.0 often recommended, though citrate buffer pH 6.0 may be used as an alternative .

What species reactivity can be expected from commonly used SHOX2 antibodies?

The species reactivity of SHOX2 antibodies varies between products:

When selecting an antibody, researchers should consider both the target species and the application. For example, antibody 16366-1-AP has demonstrated positive Western blot detection in MCF-7 cells (human), Raji cells (human), and PC-12 cells (rat), while showing positive IHC results in rat brain tissue . Cross-reactivity predictions based on sequence homology should be validated experimentally, as actual performance may vary .

How should I optimize Western blotting protocols when using SHOX2 antibodies?

Optimizing Western blotting for SHOX2 detection requires attention to several key factors:

Sample Preparation:

• When using cell lines, positive detection has been reported in MCF-7, Raji, and PC-12 cells .

• The predicted molecular weight of SHOX2 is approximately 34 kDa, but the observed molecular weight typically falls between 35-40 kDa .

Antibody Dilution:

• For rabbit polyclonal antibodies like 16366-1-AP, a dilution range of 1:200-1:1000 is recommended .

• Optimization through titration is advised for each specific experimental system to obtain optimal results .

Detection Methods:

• When using ab55740, researchers have successfully detected bands in recombinant SHOX2 protein samples and PC-12 cell lysates at 1μg/lane (with 25μg/lane of lysate) .

Controls:

• Include positive controls such as recombinant SHOX2 protein .

• For validation studies, consider using SHOX2 knockdown samples as negative controls, as referenced in published knockdown studies .

It is important to note that different versions of antibodies (such as ascites versus purified) may affect sensitivity and specificity, as mentioned in the data for ab55740 .

What are the best practices for immunohistochemistry with SHOX2 antibodies?

For optimal immunohistochemistry results with SHOX2 antibodies:

Antigen Retrieval:

• TE buffer at pH 9.0 is suggested as the primary antigen retrieval method .

• Alternatively, citrate buffer at pH 6.0 may be used, though comparative efficacy should be evaluated for specific tissues .

Antibody Dilution:

• For rabbit polyclonal antibodies like 16366-1-AP, a dilution range of 1:250-1:1000 is recommended .

• Titration is advised to determine optimal concentration for specific tissue types.

Positive Control Tissues:

• Rat brain tissue has been validated as a positive control for SHOX2 IHC .

• Consider tissues with known SHOX2 expression patterns, such as cardiac tissues (particularly focusing on pacemaker regions), developing limb structures, or adipose tissues based on research interests .

Detection Systems:

• Secondary antibody selection should match the host species of the primary antibody (rabbit for 16366-1-AP, mouse for ab55740) .

• Signal amplification methods may be necessary for low-expression tissues.

Specificity Verification:

• Consider parallel staining with multiple SHOX2 antibodies targeting different epitopes to confirm specificity .

• Include SHOX2 knockdown or knockout controls when available.

How do SHOX2 expression patterns differ between tissue types?

SHOX2 exhibits distinct expression patterns across different tissues:

Adipose Tissue:

• Higher expression in subcutaneous compared to visceral fat in both rodents and humans .

• Expression levels are further increased in humans with visceral obesity .

Cardiac Tissue:

• Expressed in fetal and adult cardiac tissues, with critical importance in pacemaker cell development .

• Influences conduction in the sinoatrial node .

Neural Tissue:

• Detected in rat brain tissue via immunohistochemistry .

• Involved in specifying neural systems for processing somatosensory information .

Skeletal System:

• Required for bone elongation and proper limb formation .

• Disruption is associated with growth retardation disorders .

Cancer Tissues:

• Elevated expression has been detected in lung cancer, colorectal cancer, and head and neck squamous cell carcinoma .

• Functions as a methylation marker for cancer detection in circulating tumor DNA and biopsy samples .

These tissue-specific expression patterns reflect SHOX2's diverse roles in development, metabolism, and pathological states.

What are the molecular interactions of SHOX2 with other transcription factors?

SHOX2 engages in significant interactions with other transcription factors to regulate gene expression:

C/EBPα Interaction:

• SHOX2 directly interacts with CCAAT/enhancer binding protein alpha (C/EBPα) and attenuates its transcriptional activity on the Adrb3 promoter .

• This interaction was confirmed through co-immunoprecipitation experiments in C3H10T1/2 cells using FLAG-tagged SHOX2 .

• The repression is dose-dependent, with SHOX2 inhibiting C/EBPα-induced activation of the Adrb3 promoter by up to 75% .

T-box Proteins:

• SHOX2 interfaces with T-box proteins, including TBX5, contributing to the specification and function of heart tissues .

• These interactions are crucial for proper formation of heart structures .

Other Potential Interactions:

• Given its role as a transcription factor in multiple developmental processes, SHOX2 likely interacts with additional transcription factors in tissue-specific contexts.

• Research into potential interactions with factors involved in adipogenesis, cardiac development, and skeletal formation is ongoing.

Understanding these molecular interactions provides insight into the mechanisms by which SHOX2 regulates gene expression in different biological contexts.

How can SHOX2 antibodies be used to study adipocyte function and metabolism?

SHOX2 antibodies offer valuable tools for investigating adipocyte biology:

Differential Expression Analysis:

• SHOX2 expression is higher in subcutaneous than visceral fat, making it a marker for depot-specific adipocyte characterization .

• Immunohistochemistry using SHOX2 antibodies can help visualize this differential expression in adipose tissue samples.

Lipolysis Regulation Studies:

• SHOX2 regulates adipocyte lipolysis by controlling β3 adrenergic receptor (Adrb3) expression .

• Co-immunoprecipitation experiments with SHOX2 antibodies can help identify protein complexes involved in this regulatory pathway.

Methodological Approach:

• Differential protein expression: Compare SHOX2 levels between visceral and subcutaneous adipose tissues using Western blot with antibodies like 16366-1-AP (1:200-1:1000) .

• Knockdown validation: When studying SHOX2 function through knockdown approaches, SHOX2 antibodies are essential for confirming reduced protein expression .

• Protein-protein interactions: Use immunoprecipitation with antibodies like sc-81955 to pull down SHOX2 and associated proteins like C/EBPα .

• Chromatin immunoprecipitation (ChIP): Employ SHOX2 antibodies in ChIP assays to identify genomic regions directly bound by SHOX2 in adipocytes, particularly in relation to genes involved in lipolysis.

These approaches provide mechanistic insights into how SHOX2 controls adipocyte-specific functions and metabolic processes.

What is the role of SHOX2 in cardiac development and how can antibodies help elucidate this function?

SHOX2 plays a crucial role in cardiac development, particularly in pacemaker formation:

Cardiac Pacemaker Function:

• SHOX2 is critical for pacemaker cell development and influences conduction in the sinoatrial node .

• Antibodies can map SHOX2 expression in fetal and adult cardiac tissues to understand developmental timing and spatial distribution.

Methodological Approaches:

• Developmental expression mapping:

  • Immunohistochemistry using antibodies like 16366-1-AP on cardiac tissues at different developmental stages .

  • Serial sections can be used to correlate SHOX2 expression with other cardiac markers.

• Protein interaction studies:

  • Co-immunoprecipitation with SHOX2 antibodies followed by mass spectrometry to identify cardiac-specific interaction partners .

  • Proximity ligation assays to visualize SHOX2 interactions with T-box proteins like TBX5 in cardiac tissue sections .

• Functional studies:

  • Western blot analysis to assess SHOX2 expression changes in models of cardiac arrhythmias or conduction disorders .

  • Immunofluorescence to examine SHOX2 localization in cardiac tissues or derived cells under normal and pathological conditions.

• Validation in model systems:

  • Use SHOX2 antibodies to confirm expression patterns in stem cell-derived cardiac pacemaker cells.

  • Compare expression in wild-type versus SHOX2 mutant cardiac tissues to understand functional consequences.

These approaches help establish the mechanistic role of SHOX2 in cardiac pacemaker development and function, potentially informing therapeutic strategies for cardiac arrhythmias.

How do epigenetic modifications of SHOX2 affect its function and detection?

Epigenetic regulation of SHOX2 has significant implications for its function and detection:

Methylation as a Biomarker:

• SHOX2 is a recognized methylation marker for cancer detection, particularly in lung cancer .

• Methylation patterns can be detected in circulating tumor DNA (ctDNA) and biopsy samples .

Impact on Antibody-Based Detection:

• Epigenetic modifications may alter protein expression levels of SHOX2, affecting antibody-based quantification.

• While antibodies detect the protein regardless of the epigenetic state of the gene, interpretation should consider how these modifications impact expression levels.

Methodological Considerations:

• Combined epigenetic and protein analysis:

  • Perform methylation-specific PCR for the SHOX2 promoter region alongside Western blot with antibodies like 16366-1-AP to correlate methylation status with protein expression .

• Chromatin state analysis:

  • Use SHOX2 antibodies in ChIP assays to examine how histone modifications correlate with SHOX2 binding to target genes.

  • Combine with ChIP-seq to identify genome-wide binding patterns under different epigenetic conditions.

• Functional studies:

  • Treat cells with epigenetic modifiers (DNMT inhibitors, HDAC inhibitors) and assess changes in SHOX2 protein levels via Western blot .

  • Examine how these treatments affect SHOX2 protein function and downstream target gene expression.

• Tissue-specific considerations:

  • Compare SHOX2 methylation patterns and protein expression across different tissues to understand tissue-specific regulation.

  • Pay particular attention to cancer vs. normal tissue comparisons, where epigenetic alterations may be most pronounced .

Understanding the relationship between SHOX2 epigenetic modifications and protein function provides insights into its role in development and disease, particularly in cancer progression.

What are the limitations and potential cross-reactivity issues with current SHOX2 antibodies?

Researchers should be aware of several limitations when working with SHOX2 antibodies:

Specificity Considerations:

• Homology with other proteins, particularly SHOX (the closely related homolog), may lead to cross-reactivity in some applications .

• Antibodies targeting different epitopes may show variable specificity and performance.

Application-Specific Limitations:

• Some antibodies may work well in certain applications but not others. For example, an antibody may be validated for Western blot but not immunohistochemistry .

• The specificity table provided by manufacturers often indicates which species/application combinations have been validated versus those predicted to work based on homology .

Validation Status:

• For 16366-1-AP, both Western blot and IHC applications have been validated in specific cell lines and tissues, but performance may vary in other contexts .

• For ab55740, validation status varies by application and species, with some combinations covered by the product promise and others not .

Methodological Approaches to Address Limitations:

• Validation controls:

  • Include SHOX2 knockdown or knockout samples as negative controls .

  • Use multiple antibodies targeting different epitopes to confirm results.

  • Include recombinant SHOX2 protein as a positive control in Western blot applications .

• Cross-reactivity testing:

  • Test antibody against recombinant SHOX to assess potential cross-reactivity with this homolog.

  • Consider pre-absorption controls to confirm specificity.

• Application optimization:

  • Titrate antibody concentrations for each specific application and sample type .

  • Test multiple antigen retrieval methods for IHC applications (TE buffer pH 9.0 vs. citrate buffer pH 6.0) .

Understanding these limitations helps researchers design appropriate controls and interpret results accurately when using SHOX2 antibodies.

How can SHOX2 antibodies be used in cancer biomarker research?

SHOX2 antibodies offer valuable tools for cancer biomarker studies:

Methylation and Expression Analysis:

• SHOX2 serves as a methylation marker for cancer detection, particularly in lung cancer .

• Antibodies can help correlate protein expression with methylation status in different cancer types.

Diagnostic and Prognostic Applications:

• Elevated SHOX2 expression has been detected in lung cancer, colorectal cancer, and head and neck squamous cell carcinoma .

• Immunohistochemistry with SHOX2 antibodies can help evaluate its potential as a diagnostic and prognostic marker.

Methodological Approaches:

• Tissue microarray analysis:

  • Use SHOX2 antibodies like 16366-1-AP (1:250-1:1000) for IHC on cancer tissue microarrays to assess expression patterns across multiple patient samples .

  • Correlate expression with clinical outcomes to evaluate prognostic value.

• Liquid biopsy studies:

  • While methylation analysis is primary for ctDNA studies, concurrent protein analysis in matched tissue samples using Western blot can provide complementary data .

• Hypoxia-related studies:

  • Recent publications mention a novel hypoxia-driven gene signature involving SHOX2 that can predict prognosis and drug resistance in gliomas .

  • Use SHOX2 antibodies to examine protein expression changes under hypoxic conditions in cancer cell lines.

• Pathway analysis:

  • Another publication mentions NRF-2/HO-1 pathway-mediated SHOX2 activation .

  • Immunoprecipitation with SHOX2 antibodies can help identify protein interactions within this pathway.

These approaches contribute to understanding SHOX2's role in cancer progression and its utility as a biomarker for various cancer types.

What are the recommended approaches for validating SHOX2 knockdown or knockout experiments?

Proper validation of SHOX2 knockdown/knockout is essential for functional studies:

Protein Level Validation:

• Western blot analysis using SHOX2 antibodies is the primary method to confirm reduced protein expression .

• For shRNA knockdown of SHOX2 in C3H10T1/2 cells, a reduction of at least 80% in SHOX2 message has been achieved and should be reflected in protein levels .

Functional Validation:

• In adipocytes, SHOX2 knockdown leads to increased expression of β3 adrenergic receptor (Adrb3) at both mRNA and protein levels, resulting in enhanced lipolysis .

• Oil Red O staining can demonstrate reduced lipid accumulation in SHOX2 knockdown cells, as observed in shSHOX2 C3H10T1/2 cells .

Methodological Approaches:

• Quantitative protein analysis:

  • Western blot with antibodies like 16366-1-AP (1:200-1:1000) or ab55740 to quantify SHOX2 protein reduction .

  • Include appropriate loading controls and quantify band intensity using image analysis software.

• Complementary techniques:

  • qPCR to confirm mRNA reduction alongside protein analysis.

  • Immunofluorescence to visualize reduced SHOX2 expression at the cellular level.

• Rescue experiments:

  • Reintroduce SHOX2 expression in knockout/knockdown models to demonstrate specificity of observed phenotypes.

  • Use SHOX2 antibodies to confirm successful re-expression.

• Downstream target analysis:

  • Measure expression of known SHOX2 targets, such as Adrb3, to confirm functional consequences of knockdown .

  • For adipocyte studies, measure lipolytic rate to confirm functional effects .

• CRISPR validation tools:

  • For CRISPR-based knockout approaches, SHOX2 CRISPR/Cas9 KO Plasmid systems are available (e.g., sc-402950) .

  • Protein-level validation with antibodies is still essential following genomic modification.

These comprehensive validation approaches ensure that observed phenotypes can be confidently attributed to SHOX2 reduction rather than off-target effects.