DUSP9 Antibody

What is DUSP9 and why is it important in cellular signaling?

DUSP9, also known as MKP-4 (Mitogen-activated protein kinase phosphatase 4), is a dual specificity phosphatase that primarily inactivates MAP kinases with specificity for the ERK family . DUSP9 plays critical roles in regulating cellular signaling pathways involved in proliferation, differentiation, and stress responses. The importance of DUSP9 lies in its ability to modulate MAPK signaling cascades, which are frequently dysregulated in various pathological conditions including cancer, cardiovascular diseases, and metabolic disorders .

The protein typically appears at molecular weights of 42-46 kDa in western blot analyses, with some variation depending on post-translational modifications and the specific antibody used . Understanding DUSP9's function provides insights into disease mechanisms and potential therapeutic targets.

Proper storage and handling of DUSP9 antibodies are crucial for maintaining their reactivity and specificity:

• Storage temperature: Store at -20°C for long-term stability .

• Aliquoting: Despite some manufacturers stating that aliquoting is unnecessary for -20°C storage , it is generally good practice to prepare small aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody performance.

• Buffer composition: Most DUSP9 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Handling precautions: Avoid contamination and exposure to strong light or heat.

• Stability: Properly stored antibodies are typically stable for one year after shipment .

Following manufacturer-specific recommendations is essential, as formulations may vary between suppliers.

What are the optimal protocols for detecting DUSP9 in Western blotting experiments?

For optimal detection of DUSP9 in Western blotting experiments, follow these methodological recommendations:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease and phosphatase inhibitors

  • Heat samples at 95°C for 5 minutes in Laemmli buffer

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels

  • Load 20-40 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF membrane (recommended over nitrocellulose)

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

• Antibody incubation:

  • Primary antibody: Dilute DUSP9 antibody 1:1000 in blocking buffer

  • Incubate overnight at 4°C

  • Secondary antibody: Use HRP-conjugated anti-rabbit at 1:3000-1:5000

  • Incubate for 1 hour at room temperature

• Detection:

  • Use ECL substrate for visualization

  • Expected molecular weight: 42-46 kDa

For validation of specificity, include a positive control (e.g., HeLa cell extract) and negative control (e.g., sample with immunizing peptide) .

How can DUSP9 expression be accurately quantified in tissue samples?

Accurate quantification of DUSP9 expression in tissue samples can be achieved through multiple complementary approaches:

• Immunohistochemistry (IHC) quantification:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections

  • DUSP9 antibody dilution: 1:50-1:200

  • Scoring method: Combine staining intensity (A) with percentage of positive cells (B)

  • Semi-quantitative analysis scale:

    • 0-1 for (-)

    • 2-4 for (+)

    • 5-8 for (++)

    • 9-12 for (+++)

• Western blotting quantification:

  • Normalize DUSP9 signal to loading controls (β-actin or GAPDH)

  • Use densitometry software (ImageJ or similar)

  • Include standard curve with recombinant protein for absolute quantification

• ELISA-based quantification:

  • Detection range: 0.313-20 ng/mL

  • Minimum detection limit: 0.313 ng/mL

  • Sample types: serum, plasma, tissue homogenates

• RT-qPCR for mRNA quantification:

  • Extract RNA using TRIzol

  • Reverse transcribe 2 μg of mRNA into cDNA

  • Perform qPCR using SYBR Green

  • Normalize to housekeeping genes

For comparative studies, it is recommended to use multiple methods to confirm findings.

What controls should be included when performing immunofluorescence with DUSP9 antibodies?

When performing immunofluorescence with DUSP9 antibodies, the following controls are essential for result validation:

• Primary antibody controls:

  • Positive control: Cell line known to express DUSP9 (e.g., HeLa cells)

  • Negative control: Omit primary antibody but include all other steps

  • Peptide competition: Incubate antibody with immunizing peptide before application

• Specificity controls:

  • siRNA/shRNA knockdown: Cells with DUSP9 expression reduced

  • Overexpression control: Cells transfected with DUSP9 expression vector

  • Isotype control: Non-specific IgG from same species as primary antibody

• Technical controls:

  • Autofluorescence control: Unstained sample to detect background

  • Secondary antibody control: Omit primary antibody to detect non-specific binding

  • Nuclear counterstain: Use DAPI to visualize nuclei and confirm cellular localization

• Quantification controls:

  • Include consistent exposure settings across all samples

  • Use proper thresholding in image analysis software

Documentation of these controls is essential for publication-quality research and troubleshooting unexpected results.

How does DUSP9 expression correlate with cancer progression and metastasis?

DUSP9 expression shows varied correlations with cancer progression depending on the cancer type, highlighting its context-dependent role:

• Colorectal cancer (CRC):

  • DUSP9 levels are significantly reduced in cancerous tissue compared to adjacent normal tissue

  • Lower DUSP9 expression correlates with enhanced proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT)

  • Functional studies demonstrate that DUSP9 inhibits CRC progression both in vitro and in vivo

  • DUSP9 may function as a tumor suppressor in CRC

• Clear cell renal carcinoma:

  • Decreased DUSP9 expression observed in cell lines and xenograft experiments

  • Loss of DUSP9 correlates with more aggressive phenotypes

  • Supports tumor suppressor role in renal cancer

• Triple-negative breast cancer:

  • Contrasting role: DUSP9 activity and expression are abnormally elevated

  • Particularly prominent in cancer stem cell-like populations

  • May contribute to tumor progression in this context

These opposing findings suggest that DUSP9's role in cancer is highly tissue-specific and dependent on the tumor microenvironment. The underlying mechanisms may involve differential regulation of MAPK pathways and interaction with tissue-specific factors.

What is the role of DUSP9 in cardiac hypertrophy and heart failure?

DUSP9 plays a protective role in cardiac hypertrophy and heart failure through specific molecular mechanisms:

• Expression patterns in cardiac stress:

  • DUSP9 is markedly upregulated in heart samples after transverse aortic constriction (TAC) surgery

  • Similar upregulation observed in cardiomyocytes challenged with angiotensin II (Ang II)

• Functional significance demonstrated through genetic models:

  • Cardiac-specific DUSP9-knockout mice show increased susceptibility to pressure overload-induced cardiac hypertrophy and malfunction

  • Cardiac DUSP9 overexpression in transgenic mice restores cardiac function and reduces hypertrophy

• Molecular mechanism:

  • DUSP9 directly interacts with and dephosphorylates ASK1 (Apoptosis Signal-regulating Kinase 1)

  • This dephosphorylation inactivates downstream p38/JNK signaling pathways

  • Absence of DUSP9 can be rescued by abolishing ASK1 in response to Ang II stimuli

• Methodological evidence:

  • Co-immunoprecipitation confirms direct physical interaction between DUSP9 and ASK1

  • Immunofluorescence demonstrates localization and interaction patterns

  • Western blot analyses show effects on phosphorylation states of signaling molecules

These findings establish DUSP9 as a novel anti-hypertrophic mediator, suggesting potential therapeutic strategies targeting DUSP9-ASK1 interaction for heart failure treatment.

How does DUSP9 function in metabolic disorders such as non-alcoholic fatty liver disease?

DUSP9 functions as a key regulator in metabolic disorders, particularly in non-alcoholic fatty liver disease (NAFLD):

• Protective role in hepatic steatosis:

  • DUSP9 acts as a key suppressor of high-fat diet-induced hepatic steatosis

  • Inhibits inflammatory responses in the liver

• Molecular mechanisms:

  • Similar to its role in cardiac tissue, DUSP9 may target ASK1 in liver tissue

  • Suppression of ASK1 and downstream signaling pathways reduces lipid accumulation and inflammation

  • Modulates MAPK signaling cascades involved in metabolic regulation

• Therapeutic implications:

  • No drugs have been approved for NAFLD and non-alcoholic steatohepatitis (NASH)

  • Therapeutics aimed at increasing DUSP9 expression in liver represent a potential treatment strategy

  • Targeting DUSP9-related pathways may ameliorate metabolic liver disorders

• Research methods for studying DUSP9 in metabolic contexts:

  • Animal models using high-fat diet challenges

  • Liver-specific DUSP9 knockout or overexpression systems

  • Primary hepatocyte cultures for mechanistic studies

  • Histological assessment of lipid accumulation and inflammation

These findings position DUSP9 as a promising therapeutic target for metabolic disorders, particularly those affecting the liver.

How does DUSP9 regulate MAPK signaling pathways and what are the downstream consequences?

DUSP9 regulation of MAPK signaling involves specific mechanisms with distinct downstream consequences:

• Substrate specificity:

  • DUSP9 primarily inactivates MAP kinases with specificity for the ERK family

  • Dephosphorylates both threonine and tyrosine residues in the activation loop of MAPKs

  • Shows higher specificity for ERK compared to p38 or JNK MAPKs

• Molecular interaction mechanisms:

  • Contains an N-terminal non-catalytic domain that determines substrate specificity

  • C-terminal catalytic domain contains the phosphatase activity

  • Forms physical complexes with target MAPKs prior to dephosphorylation

• Pathway-specific regulation:

  • In cardiac tissue: Directly dephosphorylates ASK1, inhibiting downstream p38/JNK activation

  • In cancer cells: Modulates ERK signaling, affecting proliferation and migration

  • In metabolic tissues: Regulates inflammatory responses through MAPK pathway inhibition

• Downstream consequences:

  • Reduction in transcription factor activation (including AP-1 and ELK-1)

  • Altered gene expression profiles affecting cell cycle, apoptosis, and differentiation

  • Cell-type specific responses determined by the cellular context

The complex interplay between DUSP9 and MAPK pathways creates a finely tuned regulatory network that can be disrupted in pathological conditions.

What protein-protein interactions have been identified for DUSP9 and how can they be studied?

Several protein-protein interactions involving DUSP9 have been identified, with specific methodologies for their study:

• Confirmed DUSP9 interaction partners:

  • ASK1 (Apoptosis Signal-regulating Kinase 1): Direct interaction confirmed by co-immunoprecipitation

  • ERK family MAPKs: Primary substrates for DUSP9 phosphatase activity

  • Potentially other members of MAPK signaling cascades

• Methodologies for studying DUSP9 interactions:

  a. Co-immunoprecipitation (Co-IP):

  • Transfect HEK293T cells with expression vectors for 24h

  • Lyse cells and immunoprecipitate with anti-DUSP9 antibody

  • Analyze precipitated complexes by western blot for interacting partners

  b. Double immunofluorescence analysis:

  • Allows visualization of co-localization between DUSP9 and potential interactors

  • Provides spatial information about interaction contexts

  c. Proximity ligation assay (PLA):

  • Detects protein interactions in situ with high sensitivity

  • Provides quantifiable data on endogenous protein interactions

  d. Pull-down assays with recombinant proteins:

  • Use GST-tagged or His-tagged DUSP9 to pull down interacting partners

  • Analyze by mass spectrometry for unbiased interaction discovery

• Validation approaches:

  • Mutagenesis of interaction domains to disrupt binding

  • In vitro binding assays with purified components

  • FRET/BRET approaches for real-time interaction monitoring

Understanding these interactions provides mechanistic insights into DUSP9 function and potential therapeutic targeting strategies.

How do post-translational modifications affect DUSP9 activity and stability?

Post-translational modifications (PTMs) significantly impact DUSP9 activity and stability through multiple mechanisms:

• Phosphorylation:

  • DUSP9 may be phosphorylated by upstream kinases in response to cellular stimuli

  • Potential phosphorylation sites include serine/threonine residues

  • Effects may include:

    • Altered catalytic activity

    • Changes in substrate specificity

    • Modified protein stability

    • Altered subcellular localization

• Ubiquitination:

  • May target DUSP9 for proteasomal degradation

  • Regulated by specific E3 ubiquitin ligases in response to cellular conditions

  • Controls DUSP9 protein levels under different physiological and pathological states

• Oxidation:

  • The catalytic cysteine residue in the phosphatase domain is susceptible to oxidation

  • Oxidative stress may temporarily or permanently inactivate DUSP9 enzymatic activity

  • Creates a redox-sensitive regulatory mechanism

• Methodologies to study DUSP9 PTMs:

  • Mass spectrometry to identify modification sites

  • Phospho-specific antibodies to detect phosphorylation events

  • Site-directed mutagenesis to create PTM-resistant variants

  • In vitro enzymatic assays with purified proteins to measure activity changes

Understanding these PTMs provides insights into how DUSP9 activity is fine-tuned in different cellular contexts and disease states.

What are common issues when detecting DUSP9 by western blotting and how can they be resolved?

Researchers commonly encounter several issues when detecting DUSP9 by western blotting, with specific troubleshooting approaches:

• Multiple bands or unexpected molecular weight:

  • Expected MW: 42-46 kDa

  • Issue: Post-translational modifications can shift apparent MW

  • Solution: Use positive controls and blocking peptides to confirm specificity

  • Additional approach: Include phosphatase treatment of lysates to eliminate phosphorylation-induced shifts

• Weak or no signal:

  • Issue: Low DUSP9 expression in sample or antibody sensitivity

  • Solutions:

    • Increase protein loading (40-60 μg)

    • Optimize antibody concentration (try 1:500 instead of 1:1000)

    • Increase antibody incubation time (overnight at 4°C)

    • Use enhanced chemiluminescence substrate systems

    • Consider immunoprecipitation before western blotting for enrichment

• High background:

  • Issue: Non-specific binding or inadequate blocking

  • Solutions:

    • Increase blocking time (2 hours instead of 1)

    • Try alternative blocking agents (BSA instead of milk)

    • Increase washing duration and number of washes

    • Optimize secondary antibody dilution (1:5000-1:10000)

• Inconsistent results between experiments:

  • Issue: Variable DUSP9 expression or technical factors

  • Solutions:

    • Standardize lysate preparation protocol

    • Use fresh samples (avoid repeated freeze-thaw)

    • Maintain consistent transfer conditions

    • Include internal loading controls

    • Consider housekeeping protein normalization issues

These troubleshooting approaches can significantly improve DUSP9 detection in western blotting experiments.

How can researchers optimize immunohistochemical detection of DUSP9 in different tissue types?

Optimizing immunohistochemical detection of DUSP9 across different tissue types requires systematic approach:

• Tissue fixation and processing optimization:

  • Fixation time: 12-24 hours in 10% neutral buffered formalin

  • Antigen retrieval methods:

    • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

    • For difficult tissues, try EDTA buffer (pH 9.0)

    • Optimize retrieval time (10-20 minutes)

• Antibody optimization by tissue type:

  • Placenta: DUSP9 shows strong expression; use 1:200 dilution

  • Liver: May require increased antibody concentration (1:50-1:100)

  • Cardiac tissue: 1:100 dilution with overnight incubation

  • Cancer tissues: May show variable expression; test range of dilutions (1:50-1:200)

• Detection system selection:

  • For tissues with lower expression: Use polymer-based detection systems

  • For quantitative analysis: DAB chromogen with standardized development time

  • For co-localization studies: Consider fluorescent secondary antibodies

• Validation and controls:

  • Positive tissue controls (placenta shows consistent DUSP9 expression)

  • Peptide competition controls to confirm specificity

  • Serial dilution of primary antibody to determine optimal concentration

  • Sample timing considerations (DUSP9 may vary with disease progression)

• Quantification approaches:

  • Semi-quantitative scoring system combining staining intensity and percentage of positive cells

  • Digital image analysis for more objective quantification

These optimization strategies should be tailored to the specific research question and tissue type under investigation.

What are the critical parameters for successful co-immunoprecipitation experiments with DUSP9 antibodies?

Successful co-immunoprecipitation (Co-IP) experiments with DUSP9 antibodies require attention to several critical parameters:

• Lysis buffer composition:

  • Use mild non-denaturing buffers to preserve protein-protein interactions

  • Recommended buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100

  • Include protease and phosphatase inhibitors to prevent degradation

  • For phosphatase studies, include phosphatase inhibitors (NaF, Na₃VO₄)

• Antibody selection and protocol optimization:

  • Antibody amount: 1:100 dilution for immunoprecipitation

  • Pre-clearing step: Incubate lysate with protein A/G beads before adding antibody

  • Binding conditions: 4°C overnight with gentle rotation

  • Washing stringency: Balance between removing non-specific binding and preserving interactions

• Bead selection and handling:

  • Protein A/G Sepharose or magnetic beads

  • Pre-block beads with BSA to reduce non-specific binding

  • Use gentle handling to avoid disrupting complexes

  • Elution conditions: Non-denaturing if maintaining activity is important

• Controls for result validation:

  • Input control: 5-10% of starting material

  • Negative control: Non-specific IgG from same species

  • Reverse Co-IP: Immunoprecipitate with antibody against interacting partner

  • Blocking peptide control: Confirm specificity of the interaction

• Detection strategies:

  • Western blot with specific antibodies against DUSP9 and potential interactors

  • For novel interactions: Mass spectrometry analysis of co-precipitated proteins

  • For phosphatase studies: Include phospho-specific antibodies against substrates

Applying these parameters will significantly improve the success rate and reliability of DUSP9 co-immunoprecipitation experiments.

How can DUSP9 expression be modulated for functional studies?

Several methodological approaches are available for modulating DUSP9 expression in functional studies:

• Genetic overexpression strategies:

  • Plasmid-based transient transfection in cell lines

  • Viral vectors (lentiviral, adenoviral) for stable expression or in vivo delivery

  • Inducible expression systems (Tet-On/Off) for temporal control

  • Tissue-specific promoters for targeted expression in animal models

• Knockdown/knockout approaches:

  • siRNA transfection for transient knockdown

  • shRNA for stable knockdown

  • CRISPR-Cas9 for complete knockout

  • Cardiac-specific DUSP9-knockout models have been developed for studying cardiovascular roles

• Pharmacological modulators:

  • Currently limited direct modulators of DUSP9

  • Indirect modulators of MAPK pathways can be used as complementary tools

  • Development of small molecule activators represents a significant research opportunity

• Validation of modulation:

  • Verify expression changes at mRNA level via RT-qPCR

  • Confirm protein level changes by western blot

  • Assess functional outcomes through appropriate assays:

    • Proliferation assays (MTS assay)

    • Migration and invasion assays

    • Signaling pathway activation (phosphorylation status)

    • In vivo phenotypic analysis

These approaches enable researchers to investigate the causal relationships between DUSP9 expression and biological outcomes in various disease models.

What are emerging technologies for studying DUSP9 localization and dynamics in live cells?

Emerging technologies are revolutionizing the study of DUSP9 localization and dynamics in live cells:

• Fluorescent protein fusion approaches:

  • DUSP9-GFP/RFP fusion proteins for real-time visualization

  • Photoactivatable or photoconvertible fluorescent proteins for pulse-chase experiments

  • FRET-based reporters to detect DUSP9-substrate interactions

  • Split-fluorescent protein complementation to visualize protein-protein interactions

• Advanced microscopy techniques:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Lattice light-sheet microscopy for extended 3D live imaging with minimal phototoxicity

  • FRAP (Fluorescence Recovery After Photobleaching) to measure DUSP9 mobility

  • Single-molecule tracking to follow individual DUSP9 molecules

• Optogenetic and chemogenetic tools:

  • Light-inducible DUSP9 activation or inhibition systems

  • Chemically-induced dimerization to control DUSP9 localization

  • Engineered allosteric switches for temporal control of DUSP9 activity

• Endogenous tagging strategies:

  • CRISPR-Cas9 knock-in of fluorescent tags at the endogenous DUSP9 locus

  • Auxin-inducible degron (AID) tags for rapid protein depletion

  • HaloTag or SNAP-tag fusions for flexible labeling options

• Biosensors for phosphatase activity:

  • FRET-based sensors to detect DUSP9 activity in real-time

  • Phosphorylation-dependent reporters to visualize substrate dephosphorylation

These technologies offer unprecedented insights into DUSP9 biology beyond static antibody-based detection methods.

What are the most promising therapeutic approaches targeting DUSP9 pathways in disease?

Several promising therapeutic approaches targeting DUSP9 pathways are emerging for various diseases:

• For cancer therapy (context-dependent strategies):

  • In cancers where DUSP9 acts as tumor suppressor (colorectal, renal):

    • DUSP9 expression restoration strategies (gene therapy)

    • Epigenetic modifiers to reverse silencing of DUSP9

    • Small molecule enhancers of DUSP9 expression or activity

  • In cancers where DUSP9 promotes progression (triple-negative breast cancer):

    • Selective DUSP9 inhibitors

    • Disruption of DUSP9-substrate interactions

• For cardiac hypertrophy and heart failure:

  • DUSP9 activators to enhance its cardioprotective effects

  • ASK1 inhibitors as downstream targets in DUSP9-deficient states

  • Combined approaches targeting multiple nodes in the DUSP9-ASK1-p38/JNK pathway

  • AAV-mediated cardiac-specific DUSP9 overexpression

• For metabolic disorders (NAFLD/NASH):

  • Liver-directed DUSP9 expression enhancers

  • Targeting of DUSP9-regulated inflammatory pathways

  • Combination with metabolic modulators for synergistic effects

• Drug development considerations:

  • Tissue-specific delivery systems to minimize off-target effects

  • Biomarkers for patient stratification (DUSP9 expression levels)

  • Consideration of context-dependent roles in different tissues

  • Potential for repurposing existing drugs that modulate DUSP9 pathways

• Preclinical research requirements:

  • Validation in multiple disease models

  • Toxicity and specificity assessments

  • Combination with standard-of-care therapies