meox1 Antibody

What is MEOX1 and why is it important in biological research?

MEOX1 (mesenchyme homeobox 1) is a mesodermal transcription factor with a calculated molecular weight of 28 kDa that plays critical roles in somitogenesis and sclerotome development. The protein, also known as KFS2, MOX1, or homeobox protein MOX-1, is primarily localized in the nucleus and cytoplasm, with enriched expression in adipose tissue, breast, and heart muscle . MEOX1 is essential for maintaining sclerotome polarity and formation of cranio-cervical joints, making it a significant research target for developmental biology, cardiovascular studies, and stem cell research . Recent studies have also implicated MEOX1 in pathological processes including neointima formation after vascular injury and potentially in acute myocardial infarction (AMI), expanding its research significance to vascular pathologies .

How do I select the appropriate MEOX1 antibody for my research application?

Selecting the appropriate MEOX1 antibody requires consideration of several factors:

• Target species compatibility: Verify the antibody's reactivity with your experimental model. Available MEOX1 antibodies show reactivity with various species including human (Hu), mouse (Ms), rat (Rt), rabbit (Rb), and others .

• Application suitability: Different antibodies are validated for specific applications such as Western Blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), or ELISA. For example, antibody 18190-1-AP is validated for WB with recommended dilutions of 1:2000-1:16000 .

• Epitope recognition: Consider whether you need an antibody targeting a specific region of MEOX1, such as the C-terminal region (e.g., ARP32018_P050) .

• Antibody format: MEOX1 antibodies are available as monoclonal or polyclonal, with various host species (rabbit, mouse) and in unconjugated or conjugated forms .

• Validation data: Examine provided validation data to ensure the antibody recognizes the expected molecular weight of 28-35 kDa in your tissue or cell type of interest .

A comprehensive comparison of multiple antibodies through pilot experiments is recommended for critical research applications.

What is the expected molecular weight of MEOX1 in Western blot applications?

MEOX1 has a calculated molecular weight of 28 kDa (from its 254 amino acid sequence), but is typically observed between 28-35 kDa in Western blot applications . This variation may result from post-translational modifications, alternative splicing, or species-specific differences. When using antibody 18190-1-AP, researchers have reported bands at both 28 kDa (the theoretical weight) and 35 kDa in experimental contexts . When performing Western blotting for MEOX1, appropriate positive controls such as mouse or rat brain and liver tissues should be included, as these have been validated as showing detectable MEOX1 expression . The molecular weight discrepancy between calculated and observed size is a common phenomenon for transcription factors and should be considered when interpreting results.

What are the validated applications for MEOX1 antibodies in research?

MEOX1 antibodies have been validated for multiple experimental applications:

The antibody dilution should be optimized for each experimental system, as recommended by manufacturers . When transitioning between different experimental models or tissues, validation experiments are strongly advised to confirm specificity and optimal working conditions.

How can I validate a MEOX1 antibody for research applications?

Rigorous validation of MEOX1 antibodies should include:

• Positive control testing: Use tissues with known MEOX1 expression, such as mouse brain, mouse liver, rat brain, and rat liver tissues, which have been confirmed to express detectable levels of MEOX1 .

• Molecular weight verification: Confirm that the antibody detects bands at the expected molecular weight range (28-35 kDa) in Western blot applications .

• Knockout/knockdown controls: Include MEOX1 knockdown samples (such as those treated with shMeox1) to confirm antibody specificity. Studies have successfully used Ad-shMeox1 treatments to reduce MEOX1 expression in experimental models .

• Cross-reactivity assessment: Test the antibody on tissues from multiple species if cross-species reactivity is claimed. Examine whether the staining pattern is consistent with the known cellular localization (nucleus and cytoplasm) .

• Application-specific validation: For immunohistochemistry or immunofluorescence, verify that the staining pattern correlates with known expression patterns in tissues. For instance, vascular injury models show time-dependent increases in MEOX1 expression in the adventitia, media, and neointima of blood vessels .

• RT-qPCR correlation: Correlate protein detection with mRNA expression using RT-qPCR with validated primers (e.g., MEOX1 forward: 5′-CTAGGGCCTTTGCTCCCACACT-3′ and reverse: 5′-GCCAAGAGACGCTGAGAAGCAGTA-3′) .

What tissue preparation methods are optimal for MEOX1 immunodetection?

Optimal tissue preparation for MEOX1 detection varies by application:

• For Western blot analysis:

  • Tissue homogenization in a buffer containing protease inhibitors

  • Protein extraction followed by quantification and denaturation

  • Loading 20-50 μg of total protein per lane

  • Transfer to PVDF or nitrocellulose membranes

  • Blocking with 5% non-fat milk or BSA

  • Primary antibody incubation at recommended dilutions (e.g., 1:2000-1:16000 for 18190-1-AP)

• For immunohistochemistry/immunofluorescence:

  • Fixation with 4% paraformaldehyde

  • Paraffin embedding or cryosectioning (10-15 μm sections)

  • Antigen retrieval (heat-induced in citrate buffer is often effective)

  • Blocking of endogenous peroxidases and non-specific binding

  • Overnight primary antibody incubation at 4°C

  • Species-appropriate secondary antibody detection systems

• For vascular tissue specifically:

  • Animal models like carotid artery injury using 2F-Forgaty are effective for studying MEOX1 expression dynamics

  • Collection at specific time points (e.g., days 1, 3, 7, and 14 post-injury) to capture temporal expression patterns

  • Double immunofluorescence staining can be used to co-localize MEOX1 with other markers (e.g., Sca-1+ progenitor cells)

How does MEOX1 expression change during vascular injury and repair?

MEOX1 exhibits a distinctive spatio-temporal expression pattern during vascular injury and repair:

• Temporal dynamics: Following carotid artery injury in rat models, MEOX1 expression increases in a time-dependent manner during neointima formation .

• Spatial progression: MEOX1 expression shows a spatial shift from predominantly adventitial expression (day 1 post-injury) to increased expression in media and neointima (by day 14 post-injury) .

• Correlation with progenitor cells: The expression pattern of MEOX1 correlates with the migration of Sca-1+ progenitor cells, which increase in the adventitial wall in a time-dependent manner, reaching peak levels on day 7 post-injury .

• Functional significance: Knockdown of MEOX1 using shRNA abolishes both the increased expression of Sca-1+ progenitor cells and their migration into the neointima, suggesting a causal role for MEOX1 in vascular repair mechanisms .

• Signaling pathway involvement: MEOX1 appears to regulate these processes through the activation of CDC42 and the SDF-1α/CXCR4 signaling axis, forming a synergistic effect with Rho/CDC42 pathways .

These findings indicate that MEOX1 functions as a key regulator of vascular repair mechanisms, potentially serving as both a biomarker and therapeutic target in vascular pathologies.

What signaling pathways interact with MEOX1 in cardiovascular contexts?

MEOX1 interacts with several key signaling pathways in cardiovascular contexts:

• Rho/CDC42 pathway: MEOX1 regulates the expression and activation of small Rho-family GTPases, including RhoA, Rac1, and CDC42. These proteins are co-expressed with Sca-1+ progenitor cells within the adventitia, media, and neointima of injured arteries. Knockdown of MEOX1 significantly reduces the expression of these GTPases, particularly CDC42 .

• SDF-1α/CXCR4 axis: MEOX1 concurrently regulates SDF-1α expression in vascular smooth muscle cells (VSMC) via CDC42 activation. Inhibition of CXCR4 by AMD3100 abolishes the effects of MEOX1 on Sca-1+ progenitor cell migration, suggesting a critical role for this signaling axis .

• TGF-β signaling pathway: Recent bioinformatics analyses have identified MEOX1 as one of the differentially expressed genes associated with the TGF-β signaling pathway in acute myocardial infarction. This suggests a potential role for MEOX1 in cardiac pathology through modulation of TGF-β signaling .

• Endothelial-mesenchymal transition (EndoMT): MEOX1 overexpression has been shown to induce EndoMT, a process implicated in cardiovascular disease progression. The expression patterns of MEOX1 in ventricular muscle tissue and endothelial cells are consistent with a role in regulating this cellular transition .

Understanding these signaling interactions provides insights into the molecular mechanisms by which MEOX1 influences cardiovascular pathophysiology and identifies potential targets for therapeutic intervention.

How can MEOX1 be targeted for functional studies in cardiovascular research?

Several approaches can be employed to manipulate MEOX1 function in cardiovascular research:

• Genetic knockdown: Adenoviral vectors expressing shRNA against MEOX1 (Ad-shMeox1) have been successfully used to reduce MEOX1 expression in vascular injury models. This approach reduced neointima formation, demonstrating the functional importance of MEOX1 in this process .

• Overexpression studies: Conversely, overexpression of MEOX1 has been shown to promote Sca-1+ progenitor cell migration in vitro, confirming its role in cell motility . MEOX1 overexpression can also induce EndoMT, relevant for studying vascular remodeling and fibrosis .

• Pharmacological modulation of downstream pathways: Targeting the SDF-1α/CXCR4 axis with inhibitors like AMD3100 or the Rho/CDC42 pathway with inhibitors like ZCL278 can help elucidate the mechanisms by which MEOX1 influences vascular repair .

• RT-qPCR quantification: MEOX1 mRNA expression can be reliably quantified using RT-qPCR with validated primer sets (forward: 5′-CTAGGGCCTTTGCTCCCACACT-3′, reverse: 5′-GCCAAGAGACGCTGAGAAGCAGTA-3′) .

• Animal models: The carotid artery injury model using 2F-Forgaty in rats provides an effective system for studying MEOX1 function in vascular repair. Samples should be collected at multiple time points (days 1, 3, 7, and 14) to capture the dynamic expression patterns .

• Co-expression analyses: Double immunofluorescence staining for MEOX1 with other markers (e.g., Sca-1, RhoA) can reveal functional associations and cellular contexts of MEOX1 activity .

These approaches, particularly when used in combination, provide powerful tools for dissecting the functional roles of MEOX1 in cardiovascular physiology and pathology.

What is the diagnostic potential of MEOX1 in cardiovascular diseases?

Recent research suggests MEOX1 may have significant diagnostic potential in cardiovascular diseases:

• Acute Myocardial Infarction (AMI) biomarker: Receiver Operating Characteristic (ROC) curve analysis demonstrated that MEOX1 has robust diagnostic performance for AMI with an Area Under the Curve (AUC) value of 0.801, suggesting good sensitivity and specificity as a potential biomarker .

• Differential expression in disease: MEOX1 shows significant differential expression in AMI compared to normal samples, further supporting its potential as a diagnostic indicator .

• Combined biomarker panels: MEOX1 could be used in combination with other markers such as SMURF1 (which showed an even higher AUC of 0.945) to improve diagnostic accuracy for cardiovascular conditions .

• Neointimal formation indicator: The temporal expression pattern of MEOX1 correlates with neointimal formation after vascular injury, suggesting its potential utility as a marker for vascular repair processes .

• Comparative advantage: Unlike SMURF1, which has been extensively studied, there is a lack of literature regarding MEOX1 in AMI, indicating a novel research frontier with significant potential for diagnostic innovation .

These findings highlight the need for further clinical validation studies to establish standardized assays for MEOX1 detection in patient samples and to determine optimal cutoff values for diagnostic applications.

How does MEOX1 function in endothelial-mesenchymal transition (EndoMT)?

MEOX1 plays a significant role in endothelial-mesenchymal transition (EndoMT), a process implicated in various cardiovascular pathologies:

• Expression pattern correlation: MEOX1 demonstrates consistent expression patterns in rat ventricular muscle tissue and endothelial cells, suggesting a functional role in these cell types .

• Induction of EndoMT: Experimental overexpression of MEOX1 has been shown to induce EndoMT, a cellular transdifferentiation process where endothelial cells acquire mesenchymal characteristics .

• TGF-β pathway interaction: MEOX1 is associated with the TGF-β signaling pathway, a key regulator of EndoMT. Bioinformatics analysis identified MEOX1 as one of the differentially expressed genes associated with this pathway in acute myocardial infarction contexts .

• Potential mechanism: While the exact molecular mechanisms remain to be fully elucidated, MEOX1 likely functions as a transcriptional regulator of genes involved in endothelial cell identity and mesenchymal transition.

• Research implications: The role of MEOX1 in EndoMT presents opportunities for therapeutic intervention in conditions characterized by pathological vascular remodeling, such as cardiac fibrosis, atherosclerosis, and pulmonary hypertension.

Further research is needed to identify the direct transcriptional targets of MEOX1 during EndoMT and to develop strategies for modulating this process in disease contexts.

What challenges exist in detecting and quantifying MEOX1 in experimental models?

Researchers face several challenges when working with MEOX1 in experimental settings:

• Molecular weight discrepancy: MEOX1 has a calculated molecular weight of 28 kDa but is often observed at 28-35 kDa in Western blot applications, creating potential confusion in band identification .

• Temporal expression dynamics: MEOX1 expression changes dramatically over time in response to stimuli (e.g., vascular injury), necessitating careful experimental timing for detection .

• Spatial expression heterogeneity: The protein shows differential expression across tissue layers (adventitia, media, neointima), requiring precise microdissection or in situ techniques for accurate localization studies .

• Antibody specificity concerns: As with many transcription factors, ensuring antibody specificity is challenging. Validation with positive and negative controls is essential .

• Cross-species variations: While MEOX1 is conserved across species, there may be differences in expression patterns, molecular interactions, and antibody epitopes between human, mouse, rat, and other model organisms .

• Low basal expression levels: In normal, uninjured tissues, MEOX1 expression may be relatively low, requiring sensitive detection methods and appropriate signal amplification .

• Integration with functional readouts: Correlating MEOX1 expression with functional outcomes remains challenging and often requires sophisticated animal models and cell-specific knockout approaches.

To address these challenges, researchers should employ multiple complementary techniques (e.g., Western blot, immunohistochemistry, RT-qPCR) and include appropriate controls in their experimental design.

What are common problems when using MEOX1 antibodies and how can they be resolved?

Researchers may encounter several common issues when working with MEOX1 antibodies:

• Multiple bands in Western blot:

  • Issue: Detection of bands at both 28 kDa and 35 kDa or additional unexpected bands.

  • Solution: Verify specificity with positive controls (mouse/rat brain and liver tissues) . Include a MEOX1 knockdown sample to confirm which bands are specific. Optimize antibody concentration and blocking conditions.

• Weak or no signal:

  • Issue: Low detection of MEOX1 despite expected expression.

  • Solution: Ensure sufficient protein loading (30-50 μg), optimize antibody dilution, extend incubation time, and consider more sensitive detection methods. For tissues with dynamic expression, verify the appropriate time point for peak expression .

• High background in immunostaining:

  • Issue: Non-specific staining obscuring specific MEOX1 signal.

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include additional washing steps, and consider using a different secondary antibody system.

• Inconsistent results between applications:

  • Issue: An antibody works for Western blot but not immunohistochemistry or vice versa.

  • Solution: Not all antibodies work across all applications. Verify the validated applications for each specific antibody and consider using application-specific antibodies .

• Species cross-reactivity issues:

  • Issue: Antibody fails to detect MEOX1 in the species of interest despite claimed reactivity.

  • Solution: Verify the exact species validation data and consider epitope conservation across species. Test alternative antibodies with confirmed reactivity in your species .

• Inconsistent staining patterns:

  • Issue: Variable or unexpected cellular localization of MEOX1.

  • Solution: Verify fixation and permeabilization protocols. Consider that MEOX1 localization may change in response to cellular conditions or stimuli .

How should MEOX1 expression data be quantified and statistically analyzed?

Proper quantification and statistical analysis of MEOX1 expression data requires:

• Western blot quantification:

  • Normalize MEOX1 band intensity to loading controls (GAPDH, β-actin, or β-tubulin)

  • Use digital image analysis software (ImageJ, Image Studio Lite) for densitometry

  • Express results as fold-change relative to control conditions

  • Include at least three biological replicates for statistical analysis

• Immunohistochemistry/immunofluorescence quantification:

  • Count positive cells as a percentage of total cells in defined regions

  • Measure staining intensity using standardized exposure settings

  • Perform analysis across multiple sections and samples

  • For spatial distribution studies (e.g., adventitia vs. media vs. neointima), quantify each region separately

• RT-qPCR quantification:

  • Use the 2^(-ΔΔCt) method for relative quantification

  • Normalize to appropriate reference genes (e.g., GAPDH)

  • Run samples in triplicate to ensure accuracy

  • Verify primer efficiency and specificity

• Statistical approaches:

  • For comparisons between two groups: Student's t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple group comparisons: One-way ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • For time-course studies: Two-way ANOVA or repeated measures ANOVA

  • Present data as mean ± standard deviation (SD) or standard error of the mean (SEM)

  • Consider p < 0.05 as statistically significant

• Diagnostic potential assessment:

  • Generate ROC curves and calculate AUC values to evaluate diagnostic performance

  • Determine sensitivity, specificity, and optimal cutoff values

  • Compare to established biomarkers when available

How can MEOX1 antibodies be integrated into multiplexed imaging approaches?

Integrating MEOX1 antibodies into multiplexed imaging approaches requires careful consideration:

• Antibody selection for multiplexing:

  • Choose MEOX1 antibodies raised in different host species than other target antibodies

  • If using antibodies from the same species, employ sequential staining with complete stripping or blocking between rounds

  • Consider using directly conjugated primary antibodies to avoid species cross-reactivity

• Multiplexed immunofluorescence strategies:

  • For co-localization studies (e.g., MEOX1 with Sca-1+ or RhoA), use antibodies with distinct fluorophores that have minimal spectral overlap

  • Include appropriate single-stain controls to confirm specificity and check for bleed-through

  • Consider tyramide signal amplification (TSA) for detecting low-abundance proteins alongside MEOX1

• Advanced multiplexing technologies:

  • Cyclic immunofluorescence (CycIF): Allows sequential staining and imaging of multiple markers

  • Mass cytometry imaging (IMC): Uses metal-tagged antibodies for highly multiplexed imaging

  • CODEX: CO-Detection by indEXing for highly multiplexed tissue imaging

• Analysis of multiplexed data:

  • Use specialized image analysis software for colocalization quantification

  • Consider machine learning approaches for pattern recognition in complex datasets

  • Analyze cellular neighborhoods to understand MEOX1's spatial relationships with other markers

• Validation approaches:

  • Confirm key findings with alternative methods (e.g., in situ hybridization for mRNA)

  • Consider single-cell approaches (scRNA-seq) to complement protein-level findings

• Practical considerations:

  • Optimize each antibody individually before attempting multiplexing

  • Test for antibody compatibility in pilot experiments

  • Include appropriate controls for autofluorescence and non-specific binding

These advanced imaging approaches can reveal the complex spatial relationships between MEOX1 and other proteins in cardiovascular tissues, providing insights into functional interactions and regulatory networks.