DUSP11 Antibody, HRP conjugated

What is DUSP11 and why is it an important research target?

DUSP11 (Dual Specificity Phosphatase 11, also known as PIR1) is an atypical member of the dual-specificity phosphatase family that plays multiple roles in cellular function. Unlike typical DUSPs that primarily target proteins, DUSP11 possesses RNA 5'-triphosphatase and diphosphatase activities, with relatively poor protein-tyrosine phosphatase activity . DUSP11 converts the 5' triphosphate of microRNA precursors to 5' monophosphate and regulates cellular noncoding RNA levels . It also binds directly to RNA and may participate in nuclear mRNA metabolism .

DUSP11 has emerged as significant in multiple research areas:

• Immune regulation (attenuates LPS-induced macrophage activation via TAK1)

• Viral pathogenesis (targeted by HIV-1 VPR protein)

• Cancer biology (prognostic biomarker in cholangiocarcinoma and potential innate immune checkpoint in lung cancer)

What applications are suitable for DUSP11 Antibody, HRP conjugated?

DUSP11 Antibody, HRP conjugated is primarily optimized for ELISA applications , though the unconjugated versions can be used for multiple techniques. Based on validated research applications, the recommended applications include:

The HRP conjugation eliminates the need for secondary antibody incubation in ELISA workflows, providing direct detection capability, which streamlines the experimental procedure and potentially reduces background signal .

What is the reactivity profile of commercially available DUSP11 antibodies?

The reactivity profiles vary across different antibody products. Based on the search results, most DUSP11 antibodies demonstrate:

When selecting an antibody for cross-species applications, validation data should be consulted, as the degree of conservation in epitope regions will determine cross-reactivity effectiveness .

How should DUSP11 Antibody, HRP conjugated be optimized for detecting low expression levels of DUSP11?

For detecting low expression levels of DUSP11 using HRP-conjugated antibodies, a systematic optimization approach is necessary:

• Sample preparation enhancement:

  • Use phosphatase inhibitors in lysis buffers to prevent DUSP11 degradation

  • Enrich nuclear fractions as DUSP11 has predominantly nuclear localization

  • Consider using immunoprecipitation with unconjugated antibody followed by HRP-detection

• Signal amplification strategies:

  • Use enhanced chemiluminescent substrates with higher sensitivity

  • Implement tyramide signal amplification (TSA) for ELISA applications

  • Increase sample concentration while maintaining antibody dilution at optimal range

• Quantitative optimization:

  • Perform titration experiments ranging from 1:500 to 1:5000 to determine optimal signal-to-noise ratio

  • Extend incubation time to 16-24 hours at 4°C for ELISA applications

  • Use low-protein binding materials to prevent loss of target

Recent studies have shown that DUSP11 expression may be dynamically regulated under certain conditions (e.g., downregulated during HIV-1 infection ), making careful sample timing and preparation critical for detection of biologically relevant expression levels.

What are the methodological considerations for studying DUSP11 phosphatase activity in cell-based assays?

Studying DUSP11 phosphatase activity requires specialized approaches due to its dual activity toward RNA and proteins:

• RNA phosphatase activity assessment:

  • Differential enzymatic digestion assays can be used to analyze 5' structures of RNAs (as demonstrated in HIV-1 infected Jurkat T cells)

  • Compare phosphorylation states of Y-RNAs or other Pol3-transcribed RNAs between wild-type and DUSP11 knockout/knockdown cells

  • Use radioactive labeling of RNA substrates followed by thin-layer chromatography to measure released phosphate

• Protein phosphatase activity measurement:

  • Monitor phosphorylation state of TAK1 using phospho-specific antibodies in control vs. DUSP11-depleted cells

  • Implement in vitro phosphatase assays using immunoprecipitated DUSP11 and synthetic phosphopeptides

  • Evaluate relative activity toward different phosphorylated amino acids (serine/threonine vs. tyrosine)

• Experimental controls:

  • Include catalytically inactive DUSP11 mutants as negative controls

  • Use specific phosphatase inhibitors to distinguish DUSP11 activity from other cellular phosphatases

  • Compare with related DUSPs to establish specificity profiles

When designing such assays, researchers should consider that DUSP11's phosphatase activity toward RNA is several orders of magnitude greater than its activity toward phospho-proteins , which may necessitate different detection sensitivities for each substrate type.

How can I validate DUSP11 antibody specificity in knockout/knockdown systems?

Rigorous validation of DUSP11 antibody specificity is critical for reliable experimental results. A comprehensive validation approach includes:

• Genetic knockout/knockdown controls:

  • Use CRISPR/Cas9-generated DUSP11 knockout cell lines (such approaches have been successfully employed in Jurkat T cells)

  • Implement siRNA or shRNA knockdown with at least two different targeting sequences

  • Rescue experiments with exogenous DUSP11 expression to confirm phenotype specificity

• Antibody validation methodology:

  • Compare multiple antibodies targeting different DUSP11 epitopes

  • Perform immunoblotting with recombinant DUSP11 protein as positive control

  • Pre-adsorption tests with immunizing peptide to demonstrate binding specificity

• Cross-reactivity assessment:

  • Test for signals in tissues/cells from other species with known sequence divergence

  • Evaluate potential cross-reactivity with closely related phosphatases (especially other atypical DUSPs)

  • Use mass spectrometry to confirm the identity of immunoprecipitated proteins

Published studies have demonstrated successful validation approaches using DUSP11 knockdown followed by Western blot analysis, where the disappearance of the 39 kDa band (the observed molecular weight of DUSP11) confirms antibody specificity .

How can I distinguish between RNA phosphatase and protein phosphatase activities of DUSP11 in my experimental system?

Distinguishing between the dual phosphatase activities of DUSP11 requires specific experimental approaches:

• Substrate-specific activity assays:

  • Measure dephosphorylation of 5'-triphosphorylated RNA substrates using techniques like differential enzymatic digestion

  • In parallel, assess protein dephosphorylation using phospho-specific antibodies against candidate protein targets like TAK1

  • Compare the kinetics and dose-dependency of both activities

• Mutational analysis approach:

  • Generate and express DUSP11 mutants with targeted mutations affecting the catalytic site

  • Some mutations may differentially affect RNA versus protein substrate specificity

  • Rescue experiments with these mutants in DUSP11-depleted cells can reveal which activity contributes to specific phenotypes

• Correlation analysis with phenotypes:

  • Implement RNase treatment to eliminate RNA-dependent effects

  • Use RNA phosphatase-specific inhibitors versus general phosphatase inhibitors

  • Correlation between phenotypic reversal and specific substrate dephosphorylation patterns

Research has shown that DUSP11's RNA phosphatase activity is significantly more potent than its protein phosphatase activity , with RNA dephosphorylation occurring at rates several orders of magnitude greater than protein dephosphorylation. This differential activity can be exploited to determine which function is relevant in specific contexts.

What factors might contribute to variability in DUSP11 detection across different tissue samples?

Several factors can influence the variability in DUSP11 detection across tissue samples:

• Biological variability factors:

  • Tissue-specific expression levels and post-translational modifications

  • Nuclear localization of DUSP11 may result in extraction-dependent variability

  • Disease state alterations (e.g., upregulation in cholangiocarcinoma compared to adjacent tissues)

  • Regulatory changes in response to inflammatory stimuli or viral infection

• Technical variability sources:

  • Fixation methods significantly affect epitope preservation (particularly important for IHC)

  • Different extraction methods may yield variable recovery of nuclear proteins

  • Antibody lot-to-lot variations can influence detection sensitivity

  • Cross-reactivity with related phosphatases may occur in certain tissues

• Standardization approaches:

  • Use of calibrated recombinant protein standards across experiments

  • Implementation of normalized scoring systems for immunohistochemistry (e.g., the 0-12 scale used in cholangiocarcinoma studies)

  • Comparison with mRNA expression data to validate protein-level findings

In cholangiocarcinoma research, for example, DUSP11 expression was evaluated using a semi-quantified IHC scoring system where the final IHC score was calculated as the score of percentage of positive-stained cells multiplied by the score of staining intensity. This standardized approach enabled meaningful comparison across patient samples .

How can DUSP11 antibodies be used to investigate its role in viral infection mechanisms?

DUSP11 antibodies are valuable tools for investigating the role of this phosphatase in viral infection mechanisms:

• HIV-1 infection studies:

  • Monitor DUSP11 protein levels during HIV-1 infection using Western blotting

  • Research has shown that HIV-1 infection triggers VPR-dependent downregulation of DUSP11 in vitro and in vivo

  • Compare wild-type HIV-1 with mutants lacking VPR to assess mechanism specificity

  • Track DUSP11 restoration following antiretroviral treatment (ART)

• Mechanistic investigation approaches:

  • Use immunoprecipitation to identify virus-induced changes in DUSP11 protein complexes

  • Implement immunofluorescence to track subcellular localization changes during infection

  • Combine with phospho-specific antibodies against potential downstream targets

• Clinical correlation methods:

  • DUSP11 protein levels in patient samples before and after antiviral therapy

  • DUSP11-related gene signatures as markers of infection status

  • Integration with viral load data to establish quantitative relationships

In HIV-1 research, DUSP11 antibodies have revealed that VPR expression is required for HIV-1-induced DUSP11 downregulation, which leads to hypertriphosphorylation of RNY1 and RNY4 and subsequent innate immune activation . This demonstrates how antibody-based detection can uncover critical host-pathogen interactions.

What approaches should be taken when using DUSP11 antibodies to evaluate its potential as a cancer biomarker?

When evaluating DUSP11 as a cancer biomarker using antibodies, several specialized approaches are recommended:

• Tissue microarray analysis:

  • Immunohistochemical staining of large cohorts with matched clinical data

  • Standardized scoring systems (e.g., multiplying percentage of positive cells by staining intensity)

  • Comparison between tumor and adjacent normal tissues

  • Correlation with tumor stage, grade, and patient outcomes

• Prognostic value assessment:

  • Kaplan-Meier survival analysis stratified by DUSP11 expression levels

  • Multivariate Cox regression to assess independent prognostic value

  • Time-dependent ROC analysis to determine optimal cutoff points

• Cancer subtype differentiation:

  • Compare expression patterns across cancer subtypes (e.g., intrahepatic vs. extrahepatic cholangiocarcinoma)

  • Integration with molecular subtyping data

  • Correlation with other established biomarkers

Research has shown that DUSP11 expression has cancer-specific and even subtype-specific prognostic value, highlighting the importance of contextualized biomarker evaluation .

How can DUSP11 antibodies be utilized to investigate its role in innate immune regulation?

DUSP11 antibodies provide critical tools for examining the role of this phosphatase in innate immune regulation:

• Immune cell signaling analysis:

  • Western blot analysis to monitor DUSP11 expression changes during immune activation

  • Co-immunoprecipitation to identify interactions with immune signaling components like TAK1

  • Phospho-specific antibody panels to track signaling pathway alterations in wild-type versus DUSP11-deficient cells

• Mechanistic investigation approaches:

  • Comparison of LPS-induced TAK1 phosphorylation between wild-type and DUSP11-knockout macrophages

  • Assessment of cytokine production patterns using combined antibody-based techniques

  • Tracking of interferon-regulated gene expression following DUSP11 manipulation

• In vivo immune response characterization:

  • Analysis of serum cytokine levels in DUSP11-deficient versus wild-type mice after immune challenge

  • Tissue-specific immunostaining to map DUSP11 expression in immune organs

  • Correlation of DUSP11 expression with immune infiltration patterns in disease models

Research has demonstrated that DUSP11 deficiency enhances LPS-induced TAK1 phosphorylation and cytokine production in bone marrow-derived macrophages, and DUSP11-deficient mice are more susceptible to LPS-induced endotoxic shock with significantly elevated serum levels of pro-inflammatory cytokines . These findings establish DUSP11 as a negative regulator of innate immune responses.

What specialized techniques can be combined with DUSP11 antibodies to investigate its RNA-regulatory functions?

To investigate DUSP11's RNA-regulatory functions, researchers can combine antibody-based techniques with specialized RNA analysis approaches:

• RNA-protein interaction analysis:

  • RNA immunoprecipitation (RIP) using DUSP11 antibodies to isolate bound RNA species

  • Crosslinking immunoprecipitation (CLIP) for higher resolution mapping of binding sites

  • Mass spectrometry of DUSP11-containing ribonucleoprotein complexes

• RNA 5' end phosphorylation analysis:

  • Differential enzymatic digestion assays to analyze 5' structure of RNAs in wild-type versus DUSP11-deficient cells

  • 5' RNA sequencing to globally profile RNA 5' end modifications

  • In vitro dephosphorylation assays using immunopurified DUSP11 and synthetic RNA substrates

• Functional consequence assessment:

  • Analysis of RNY4 and other Y-RNA processing in the context of DUSP11 manipulation

  • Integration with RNA stability and expression profiling data

  • RNA silencing assays to evaluate the impact of DUSP11 on RNA regulatory pathways

Research has established that DUSP11 functions as an RNA 5'-triphosphatase that regulates the phosphorylation state of various cellular noncoding RNAs . During HIV-1 infection, VPR-mediated degradation of DUSP11 leads to hypertriphosphorylation of RNY1 and RNY4, contributing to innate immune activation . These observations highlight the importance of combining antibody-based protein detection with specialized RNA analysis techniques.

What are common challenges in Western blot optimization for DUSP11 detection and how can they be addressed?

Several challenges can arise when optimizing Western blots for DUSP11 detection:

• Band specificity issues:

  • Challenge: Multiple bands or non-specific binding

  • Solution: Increase blocking time/concentration, optimize antibody dilution (1:2000-1:12000 range is recommended ), and include appropriate knockout/knockdown controls

  • Validate with multiple antibodies targeting different epitopes of DUSP11

• Low signal intensity:

  • Challenge: Weak detection of the 39 kDa DUSP11 band

  • Solution: Optimize protein extraction (particularly for nuclear proteins), increase protein loading (50-100 μg), extend primary antibody incubation time (overnight at 4°C), and use enhanced chemiluminescence detection

  • Consider using signal enhancers or amplification systems

• Variable expression levels:

  • Challenge: Inconsistent DUSP11 detection across samples

  • Solution: Consider cell/tissue-specific expression patterns, standardize sample collection timing, and include appropriate loading controls

  • Be aware that certain conditions (like viral infection) can dramatically alter DUSP11 levels

Researchers should be aware that DUSP11 has a predominantly nuclear localization , which may necessitate specialized extraction protocols for optimal detection.

How can I optimize immunohistochemical detection of DUSP11 in different tissue types?

Optimizing immunohistochemical detection of DUSP11 across diverse tissues requires consideration of several factors:

• Tissue fixation and processing optimization:

  • Standardize fixation time (10% neutral buffered formalin for 24-48 hours)

  • Optimize antigen retrieval methods (citrate buffer pH 6.0 has been successfully used)

  • Consider tissue-specific modifications based on cellular density and composition

• Antibody optimization strategies:

  • Perform antibody titration experiments (typically starting with 1:100-1:500 dilutions)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use detection amplification systems for tissues with low expression

• Tissue-specific considerations:

  • Include positive control tissues with known DUSP11 expression

  • Implement appropriate negative controls (both technical and biological)

  • Consider tissue-specific blocking reagents to minimize background

In cholangiocarcinoma research, successful DUSP11 IHC protocols include deparaffinization and rehydration with xylene and graded alcohol, 3% hydrogen peroxide treatment, citrate buffer antigen retrieval (pH 6.0), and blocking with 1% bovine serum albumin. Primary antibody incubation is performed overnight at 4°C, followed by secondary antibody incubation for 1 hour and visualization with 3,3'-diaminobenzidine .

The semi-quantification of DUSP11 expression can be accomplished using a scoring system that combines staining intensity (0-3) with percentage of positive cells (1-4), yielding a final score range of 0-12 .

How might DUSP11 antibodies be used to investigate its role as an innate immune checkpoint in cancer therapy?

Recent research has identified DUSP11 as a potential innate immune checkpoint in cancer, particularly in Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma . Antibody-based techniques can facilitate several investigative approaches:

• Mechanistic investigation strategies:

  • Western blot analysis to correlate DUSP11 expression with immune evasion markers

  • Immunoprecipitation to identify interactions with pattern recognition receptors like RIG-I

  • Immunohistochemistry to co-localize DUSP11 with immune cell infiltrates in tumor tissues

• Therapeutic response monitoring:

  • Quantitative assessment of DUSP11 expression changes during immunotherapy

  • Correlation of baseline DUSP11 levels with response to immune checkpoint inhibitors

  • Analysis of DUSP11 knockdown effects on tumor cell susceptibility to immune attack

• Translational research applications:

  • Development of companion diagnostic assays using standardized IHC protocols

  • Screening for small molecule inhibitors of DUSP11 using antibody-based activity assays

  • Combination studies with established immune checkpoint inhibitors

Recent findings indicate that DUSP11 knockdown in lung adenocarcinoma cells induces apoptosis and an innate immune response capable of activating other cells in vitro, with evidence suggesting that these phenotypes are primarily mediated by the pattern recognition receptor RIG-I . Furthermore, DUSP11 expression appears to be important for tumor engraftment and growth of human lung adenocarcinoma in mice, positioning it as a promising therapeutic target .

What are the potential applications of studying DUSP11 in neurodegenerative disorders?

While direct evidence linking DUSP11 to neurodegenerative disorders is limited, several characteristics of this phosphatase suggest potential relevance that could be investigated using antibody-based approaches:

• RNA metabolism connections:

  • DUSP11's role in RNA processing may be relevant to RNA-based pathologies in neurodegeneration

  • Immunohistochemical analysis of DUSP11 expression in patient brain tissues

  • Co-localization studies with RNA stress granules or other RNA-processing bodies

• Inflammatory signaling regulation:

  • Given DUSP11's role in attenuating inflammatory responses , it may modulate neuroinflammation

  • Analysis of DUSP11 expression in activated microglia or astrocytes

  • Correlation between DUSP11 levels and inflammatory markers in neurodegenerative tissues

• Phosphorylation regulation:

  • DUSP11's ability to regulate TAK1 signaling might influence neuroinflammatory cascades

  • Investigation of potential protein substrates relevant to neurodegeneration

  • Tracking phosphorylation changes in disease-relevant proteins following DUSP11 manipulation

These applications would require validated antibodies for brain tissue analysis, optimization of neuron-specific extraction protocols, and careful correlation with disease-specific markers. While speculative, the exploration of DUSP11 in neurodegenerative contexts represents a novel research direction worthy of investigation.

How can DUSP11 antibodies contribute to understanding its broader role in RNA metabolism across different cellular contexts?

DUSP11 antibodies can facilitate comprehensive investigation of its RNA metabolism functions across diverse cellular contexts:

• Global RNA processing analysis:

  • Immunoprecipitation of DUSP11-containing complexes followed by RNA sequencing

  • ChIP-seq approaches to map DUSP11 association with chromatin at sites of active transcription

  • Correlation of DUSP11 levels with global changes in RNA 5' end modifications

• Cell type-specific studies:

  • Immunohistochemical mapping of DUSP11 expression across tissues and cell types

  • Analysis of RNA substrate preferences in different cellular contexts

  • Correlation of DUSP11 levels with cell type-specific RNA processing events

• Stress response investigations:

  • Monitoring DUSP11 localization and expression during cellular stress conditions

  • Co-localization with stress granules, P-bodies, or other RNA processing centers

  • Correlation with stress-induced changes in RNA processing and stability