UTY Antibody

What is UTY and what are its primary biological functions?

UTY is a protein encoded by the Y chromosome-specific gene that functions as an epigenetic regulator. Unlike its X-chromosome paralog UTX, UTY demonstrates distinctive functional properties while maintaining structural similarities. UTY plays crucial roles in gene regulation by interacting with members of the Groucho/transducin-like Enhancer of split (TLE) family, which are vertebrate orthologs of the yeast Tup1 protein . This interaction facilitates the formation of transcriptional repressor complexes that modulate gene expression across various biological processes.

Recent research has revealed UTY's protective functions in several disease contexts. For instance, UTY helps protect against pulmonary hypertension by regulating the expression of proinflammatory chemokines CXCL9 and CXCL10 . Additionally, UTY disruption in leukocytes has been linked to accelerated progression of heart failure in male mice with loss of Y chromosome (LOY), demonstrating its importance in cardiovascular health .

The gene is expressed in most tissues in male mice , suggesting widespread physiological relevance that extends beyond reproductive functions typically associated with Y-chromosome genes.

How do commercially available UTY antibodies differ in their applications and specificities?

Different UTY antibodies vary significantly in their target epitopes, host species, and validated applications, which directly impacts their research utility. Based on current commercial offerings, researchers should consider these key differences when selecting an appropriate antibody:

When selecting an antibody, researchers should carefully consider experimental requirements. For example, monoclonal antibodies like UTY (E4X6V) provide excellent specificity and lot-to-lot consistency, while polyclonal antibodies may offer enhanced sensitivity by recognizing multiple epitopes .

A critical methodological consideration is that many UTY antibodies may cross-react with UTX due to sequence homology. As noted in research literature, "UTY antibodies likely detected ChrX paralog UTX, as they revealed a signal in females" . This highlights the importance of proper validation when interpreting results.

What validation methods should be employed to confirm UTY antibody specificity?

Ensuring antibody specificity is paramount for obtaining reliable data in UTY research. Recommended validation approaches include:

• Genetic controls: Compare antibody signals between male (UTY-positive) and female (UTY-negative) samples. Absence of signal in female samples strongly supports specificity for UTY over UTX.

• Knockdown/knockout validation: Employ UTY-knockdown models to confirm signal reduction. As demonstrated in pulmonary hypertension research, "Knockdown of the Y-chromosome gene Uty resulted in more severe PH" and allowed for verification of antibody specificity .

• RNAscope validation: As noted in pulmonary hypertension studies, "RNAscope in situ probes were used for imaging after we determined that UTY antibodies likely detected ChrX paralog UTX." This approach has been described as producing "the first to depict specific Uty expression in mouse and human lung tissue" .

• Western blot analysis: Confirm detection of appropriately sized bands (approximately 135 kDa for UTY) and absence in female samples.

• Immunoprecipitation followed by mass spectrometry: For definitive identification of the target protein.

What are the optimal protocols for using UTY antibodies in Western blotting?

Western blotting represents one of the primary applications for UTY antibodies. Based on protocol recommendations and research practices, the following methodology is suggested:

• Sample preparation:

  • Prepare tissue/cell lysates in RIPA buffer supplemented with protease inhibitors

  • Aim for 30-50 μg of total protein per lane for endogenous UTY detection

  • Include male and female samples as positive and negative controls, respectively

• Gel electrophoresis:

  • Use 8% SDS-PAGE gels due to UTY's high molecular weight (135 kDa)

  • Run at lower voltage (80-100V) to improve resolution of high molecular weight proteins

• Transfer and blocking:

  • Employ wet transfer for 2 hours at 100V or overnight at 30V (4°C) for efficient transfer of large proteins

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • For UTY (E4X6V) Rabbit mAb: Dilute 1:1000 in 5% BSA in TBST

  • For Anti-UTY (C-term): Follow manufacturer's recommended dilution

  • Incubate overnight at 4°C with gentle agitation

• Detection and visualization:

  • Use appropriate secondary antibodies conjugated to HRP (typically 1:2000-1:5000)

  • Develop using enhanced chemiluminescence

  • Expected molecular weight for UTY is approximately 135 kDa

Troubleshooting tip: If background is high, increase washing steps or reduce primary antibody concentration. If signal is weak, consider longer exposure times or signal amplification methods.

How can UTY antibodies be effectively employed in immunofluorescence studies?

Immunofluorescence (IF) studies require specific optimization for successful UTY visualization. Based on available protocols and research applications:

• Sample preparation:

  • For cultured cells: Fix with 4% paraformaldehyde for 15 minutes, permeabilize with 0.1% Triton X-100

  • For tissue sections: Use 5-7 μm sections, deparaffinize and perform antigen retrieval (citrate buffer pH 6.0)

• Blocking and antibody incubation:

  • Block with 5-10% normal serum (matching secondary antibody host) in PBS with 0.1% Triton X-100

  • Incubate with primary antibody at manufacturer-recommended dilution (typically 1:100-1:500) overnight at 4°C

  • Wash thoroughly and incubate with fluorophore-conjugated secondary antibody (1:500-1:1000) for 1-2 hours

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Mount with anti-fade mounting medium

• Controls and validation:

  • Include male and female samples

  • Consider dual staining with cell-type specific markers to identify UTY-expressing cell populations

As demonstrated in published research, confocal immunofluorescent analysis with UTY antibodies can be performed followed by visualization with appropriate secondary antibodies (e.g., "Alexa Fluor 488-conjugated goat anti-rabbit IgG"). Actin filaments can be co-stained with "Alexa Fluor 555 phalloidin" and nuclei with DAPI .

What approaches can be used to study UTY in tissue-specific contexts?

Investigating UTY in tissue-specific contexts requires specialized approaches due to its variable expression patterns and functional roles across tissues:

• RNAscope in situ hybridization:

  • This technique has proven valuable for specific detection of UTY mRNA in tissues

  • Research indicates this approach produces "the first to depict specific Uty expression in mouse and human lung tissue"

  • Allows co-localization studies with other markers (e.g., "colocalization of Uty with Cxcl9 and Cxcl10 in lung macrophages")

• Immunohistochemistry with tissue-specific validation:

  • Optimize antigen retrieval methods based on tissue type

  • Include appropriate tissue-specific controls

  • Consider dual staining with cell lineage markers

• Tissue-specific knockout models:

  • Generate conditional Uty knockout models using tissue-specific Cre recombinase expression

  • As demonstrated in research: "macrophage-specific Uty-KD impairs immune activation"

• Flow cytometry for immune cell analysis:

  • Utilize UTY antibodies compatible with flow cytometry (e.g., UTY Antibody (G-3) FITC)

  • Allows quantification of UTY expression across different immune cell populations

  • Research has shown successful "flow cytometric analysis of CEM cells" using UTY antibodies

• Single-cell RNA sequencing:

  • Provides high-resolution analysis of UTY expression patterns

  • Can reveal cell type-specific functions of UTY across tissues

How can UTY antibodies be utilized to investigate epigenetic regulation mechanisms?

UTY functions as an epigenetic regulator with histone demethylase activity, though with lower catalytic efficiency than its paralog UTX. Research approaches to investigate UTY's epigenetic functions include:

• Chromatin immunoprecipitation (ChIP):

  • Use UTY antibodies to identify genomic regions bound by UTY

  • Protocol adjustments: Increase chromatin amount (4-5x standard), extend antibody incubation time (overnight)

  • Follow with sequencing (ChIP-seq) to map UTY binding sites genome-wide

• Co-immunoprecipitation (Co-IP):

  • Utilize UTY antibodies for immunoprecipitation (e.g., UTY (E4X6V) Rabbit mAb at 1:50 dilution)

  • Identify UTY-interacting proteins that form part of epigenetic complexes

  • Research has shown that "Both UTX and JMJD3 interact with mixed-lineage leukemia (MLL) complexes 2 and 3"

• Sequential ChIP (Re-ChIP):

  • Perform sequential immunoprecipitation with UTY antibodies and antibodies against histone modifications

  • Maps relationship between UTY binding and specific histone modification patterns

• Histone modification analyses:

  • Compare histone modification profiles (particularly H3K27me3) between UTY-expressing and UTY-deficient cells

  • Can be performed using ChIP-seq or mass spectrometry approaches

• Transcriptional reporter assays:

  • Assess UTY's impact on gene expression using reporter constructs

  • Evaluate UTY's interaction with TLE family members in transcriptional repression

Recent research demonstrates that "UTY functions to regulate HOX gene expression during development" , making developmental gene loci particular targets of interest for epigenetic studies.

What role does UTY play in cardiovascular pathologies and how can this be studied?

UTY has emerged as a significant factor in cardiovascular disease, with specific protective functions. Research approaches to investigate these roles include:

• Heart failure models with UTY manipulation:

  • Research has shown that "Uty disruption leads to epigenetic alterations in both monocytes and macrophages, increasing the propensity of differentiation into profibrotic macrophages"

  • Experimental approach: Generate UTY-deficient leukocyte models and assess cardiac function

• Pulmonary hypertension models:

  • Studies demonstrate that "Knockdown of the Y-chromosome gene Uty resulted in more severe PH measured by increased right ventricular pressure and decreased pulmonary artery acceleration time"

  • Methodological approach: UTY knockdown followed by hypoxia exposure and hemodynamic measurements

• Inflammatory cytokine analysis:

  • UTY regulates pro-inflammatory chemokines: "RNA sequencing revealed an increase in proinflammatory chemokines Cxcl9 and Cxcl10 as a result of Uty knockdown"

  • Analytical approach: Cytokine profiling in UTY-deficient versus control conditions

• Therapeutic intervention studies:

  • Research demonstrates that "Treatment with a transforming growth factor-β-neutralizing antibody prevented the cardiac pathology associated with Uty deficiency in leukocytes"

  • Experimental design: Test candidate compounds in UTY-deficient cardiovascular disease models

• Sex-specific comparative analyses:

  • Investigate why "CXCL9 and CXCL10 [are] significantly upregulated in human PAH lungs, with more robust upregulation in females with PAH"

  • Approach: Comparative transcriptomics and epigenomics between male and female samples

These methodologies collectively provide a comprehensive framework for investigating UTY's cardiovascular roles, potentially leading to sex-specific therapeutic approaches.

How can RNAscope be combined with UTY antibodies for comprehensive protein-transcript analyses?

Combining RNAscope in situ hybridization with immunofluorescence using UTY antibodies offers powerful insights into UTY biology:

• Sequential RNAscope and immunofluorescence protocol:

  • First perform RNAscope for UTY transcript detection

  • Follow with immunofluorescence using UTY antibodies

  • This approach allows direct correlation between mRNA and protein expression

• Validation and specificity considerations:

  • As noted in research, "RNAscope in situ probes were used for imaging after we determined that UTY antibodies likely detected ChrX paralog UTX"

  • This combined approach helps overcome antibody specificity limitations

• Multi-parameter analysis:

  • Include additional probes and antibodies against interacting partners or downstream targets

  • Example: "We confirmed colocalization of Uty with Cxcl9 and Cxcl10 in lung macrophages using RNAscope in situ hybridization"

• Single-cell resolution analysis:

  • Apply to tissue sections to maintain spatial context

  • Quantify at single-cell level to identify heterogeneity in UTY expression and function

• Three-dimensional reconstruction:

  • Perform on serial sections for 3D mapping of UTY expression patterns

  • Particularly valuable for complex tissues like brain or developing embryos

What are common challenges in UTY antibody applications and how can they be addressed?

Researchers frequently encounter specific challenges when working with UTY antibodies. Here are evidence-based solutions:

• Cross-reactivity with UTX:

  • Problem: "UTY antibodies likely detected ChrX paralog UTX, as they revealed a signal in females"

  • Solution: Always include female samples as negative controls; consider RNAscope for validation; use Y-chromosome-specific genetic approaches

• Low signal intensity:

  • Problem: UTY's relatively low expression levels can result in weak detection

  • Solution: Increase primary antibody concentration; extend incubation times; employ signal amplification methods; increase protein loading for Western blots

• Non-specific banding patterns:

  • Problem: Multiple bands appearing in Western blot

  • Solution: Optimize blocking conditions (5% milk vs. BSA); titrate antibody concentration; increase washing stringency; verify expected molecular weight (135 kDa for UTY)

• Tissue-specific optimization requirements:

  • Problem: Standard protocols may not work across all tissue types

  • Solution: Optimize fixation methods and antigen retrieval conditions for each tissue type; consider tissue-specific positive controls

• Lot-to-lot variability:

  • Problem: Inconsistent results between antibody lots, particularly with polyclonal antibodies

  • Solution: Consider recombinant monoclonal antibodies for "Superior lot-to-lot consistency, continuous supply, and animal-free manufacturing" ; validate each new lot against previous results

What quality control measures are essential when using UTY antibodies?

Implementing rigorous quality control is essential for reliable UTY research:

• Genetic validation controls:

  • Male vs. female samples (UTY is Y-chromosome encoded)

  • UTY knockout/knockdown samples when available

  • These controls provide definitive validation of specificity

• Application-specific controls:

  • For Western blotting: Molecular weight markers to confirm expected size (135 kDa)

  • For immunofluorescence: Secondary-only controls to assess background

  • For flow cytometry: Isotype controls and fluorescence-minus-one (FMO) controls

• Cross-validation between techniques:

  • Verify findings using orthogonal methods (e.g., RNAscope plus protein detection)

  • As demonstrated in research: "We confirmed colocalization of Uty with Cxcl9 and Cxcl10 in lung macrophages using RNAscope in situ hybridization"

• Antibody validation documentation:

  • Review manufacturer's validation data

  • Document in-house validation experiments

  • Consider publishing validation protocols as supplementary methods

• Replication across experimental conditions:

  • Test antibody performance across different fixation methods

  • Validate across multiple biological replicates

  • Ensure reproducibility between researchers in the same laboratory

How is UTY being investigated in inflammatory and immune-related disorders?

UTY plays significant roles in immune regulation with implications for various disorders:

• Macrophage polarization and function:

  • Research demonstrates that "Uty disruption leads to epigenetic alterations in both monocytes and macrophages, increasing the propensity of differentiation into profibrotic macrophages"

  • "Macrophage-specific Uty-KD impairs immune activation"

  • Future research directions: Investigate UTY's role in macrophage polarization across different disease contexts

• Chemokine regulation:

  • "RNA sequencing revealed an increase in proinflammatory chemokines Cxcl9 and Cxcl10 as a result of Uty knockdown"

  • Research application: Use UTY antibodies to identify direct genomic targets involved in chemokine regulation

• Sex differences in inflammatory responses:

  • "CXCL9 and CXCL10 [are] significantly upregulated in human PAH lungs, with more robust upregulation in females with PAH"

  • Emerging research area: Sex-specific inflammatory signatures and their relationship to UTY expression

• Therapeutic interventions targeting UTY-regulated pathways:

  • "Inhibition of Cxcl9 and Cxcl10 expression in male Uty knockout mice and CXCL9 and CXCL10 activity in female rats significantly reduced PH severity"

  • Translational opportunity: Develop therapeutic strategies targeting UTY-regulated inflammatory pathways

• Atherosclerosis research:

  • "UTY expression, specifically in macrophages, is associated with increased atherosclerosis risk in men"

  • Future direction: Investigate UTY's role in sex-specific cardiovascular disease risk

What emerging technologies are enhancing UTY research beyond conventional antibody applications?

Beyond traditional antibody applications, cutting-edge technologies are advancing UTY research:

• CRISPR-based approaches:

  • CRISPR-Cas9 for precise UTY gene editing

  • CRISPRi/CRISPRa for modulating UTY expression without genetic modification

  • CRISPR-based tagging of endogenous UTY protein (avoiding antibody limitations)

• Single-cell multi-omics:

  • Combined single-cell transcriptomics and proteomics to correlate UTY transcript and protein levels

  • Single-cell epigenomics to map UTY's impact on chromatin state

  • Spatial transcriptomics to map UTY expression patterns with tissue context

• Proteomics approaches:

  • Proximity labeling methods (BioID, APEX) to identify UTY-interacting proteins

  • Targeted proteomics for precise quantification of UTY and its post-translational modifications

  • Cross-linking mass spectrometry to map UTY protein structure and interactions

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed subcellular localization of UTY

  • Live-cell imaging of tagged UTY to track dynamic processes

  • Tissue clearing methods combined with immunolabeling for whole-organ UTY mapping

• Computational biology integration:

  • Machine learning approaches to predict UTY binding sites and regulatory networks

  • Systems biology modeling of UTY-dependent pathways

  • Comparative genomics to understand evolutionary conservation of UTY function