DUSP13 Antibody, FITC conjugated

What is DUSP13 and what is its biological significance?

Dual Specificity Phosphatase 13 (DUSP13) is a member of the protein tyrosine phosphatase (PTP) family that functions as a regulator of cellular signaling pathways. DUSP13 exists in two isoforms: DUSP13A and DUSP13B, which are encoded by the same gene but from different open reading frames. DUSP13A has been identified as a novel regulator of Apoptosis Signal-regulating Kinase 1 (ASK1), a member of the MAP kinase kinase kinase family that plays a crucial role in stress-induced cell death pathways .

DUSP13A specifically interacts with the N-terminal domain of ASK1 and enhances ASK1-mediated apoptosis by activating downstream factors, including the caspase cascade. Notably, DUSP13A can induce ASK1-mediated cell death through the activation of caspase-3 and caspase-9, leading to cytochrome c release from mitochondria . This regulatory function positions DUSP13 as an important player in cellular stress response and programmed cell death mechanisms.

What are the common applications for DUSP13 antibodies in research?

DUSP13 antibodies are valuable tools for investigating the expression, localization, and function of DUSP13 in various experimental systems. Based on available data, common applications include:

• Western Blotting (WB): For detecting and quantifying DUSP13 protein expression in cell or tissue lysates .

• Immunofluorescence (IF): Both for cultured cells (IF-cc) and paraffin-embedded tissue sections (IF-p), allowing visualization of DUSP13 localization within cellular compartments .

• Co-immunoprecipitation (Co-IP) assays: For studying protein-protein interactions, particularly between DUSP13A and binding partners such as ASK1 .

• Kinase activity assays: For investigating the effect of DUSP13A on ASK1 kinase activity and downstream signaling events .

• Cell death and apoptosis assays: For assessing the functional role of DUSP13A in regulating programmed cell death pathways .

The selection of appropriate application depends on the specific research question, available samples, and experimental design considerations.

How do I select the appropriate DUSP13 antibody for my experiment?

Selecting the appropriate DUSP13 antibody requires consideration of several important factors:

• Target specificity: Determine which isoform of DUSP13 (DUSP13A or DUSP13B) is relevant to your research. Some antibodies are specific to particular amino acid sequences, such as those binding to AA 81-198 or AA 147-173 regions .

• Host species: Consider compatibility with your experimental system. Rabbit-derived polyclonal antibodies are commonly available for DUSP13 .

• Application compatibility: Verify that the antibody has been validated for your intended application (WB, IF, IHC, etc.) .

• Conjugation requirements: Determine if you need an unconjugated antibody or one conjugated to a fluorophore like FITC or AbBy Fluor® 594, depending on your detection method .

• Species reactivity: Check if the antibody recognizes DUSP13 from your species of interest. Some antibodies react with human DUSP13 only, while others can detect mouse, rat, cow, or sheep variants as well .

• Epitope targeting: Consider whether you need an antibody targeting the N-terminal region, C-terminal region, or other specific domains of DUSP13 .

For studying DUSP13's role in ASK1 regulation specifically, antibodies recognizing the N-terminal domain interaction sites would be particularly valuable for mechanistic studies .

How does DUSP13A regulate ASK1 activity in relation to apoptotic pathways?

DUSP13A exhibits a sophisticated mechanism for regulating ASK1 activity in apoptotic pathways that extends beyond conventional phosphatase activity. Research has revealed several key aspects of this regulation:

• Direct binding and activation: DUSP13A interacts with the N-terminal domain (residues 1-666) of ASK1 and enhances its kinase activity in a dose-dependent manner. This interaction leads to increased ASK1 autophosphorylation and subsequent activation of downstream targets .

• Competition with Akt1: DUSP13A competes with Akt1, a negative regulator of ASK1, for binding to ASK1. This competition prevents Akt1-mediated phosphorylation of ASK1 at Ser-83, a modification that inhibits ASK1 activity. Through in vitro binding competition assays, it has been demonstrated that increasing levels of DUSP13A (either wild-type or phosphatase-inactive mutant) progressively reduce Akt1 binding to ASK1 .

• Phosphatase-independent mechanism: Interestingly, the catalytically inactive DUSP13A mutant (DACS) is equally effective as the wild-type protein in activating ASK1. This indicates that DUSP13A's regulatory function on ASK1 is independent of its phosphatase activity .

• Enhancement of apoptotic signaling: DUSP13A enhances ASK1-mediated activation of downstream factors including JNK and p38 MAPK pathways. This leads to increased caspase-3 and caspase-9 activation and promotes cytochrome c release from mitochondria, culminating in apoptotic cell death .

These findings highlight DUSP13A as a positive regulator of ASK1-mediated apoptosis through a mechanism primarily involving protein-protein interactions rather than enzymatic dephosphorylation.

What are the technical considerations when using FITC-conjugated DUSP13 antibodies for cellular localization studies?

When employing FITC-conjugated DUSP13 antibodies for cellular localization studies, researchers should consider several technical aspects to ensure accurate and reproducible results:

• Spectral properties and photobleaching: FITC (fluorescein isothiocyanate) has excitation/emission maxima around 495/519 nm. This fluorophore is susceptible to photobleaching, so minimize exposure to excitation light and consider using anti-fade mounting media containing anti-photobleaching agents .

• Autofluorescence management: Cellular components like NADH, flavins, and lipofuscin can generate autofluorescence in the same spectral range as FITC. Consider using appropriate controls and background subtraction techniques to differentiate specific DUSP13 signal from autofluorescence .

• Fixation and permeabilization optimization: Different fixation methods (paraformaldehyde, methanol, etc.) and permeabilization agents can affect antibody access to DUSP13 epitopes and FITC fluorescence intensity. Optimization for your specific cell type is essential .

• Subcellular compartmentalization analysis: Since DUSP13A has been shown to interact with ASK1 and influence mitochondrial-mediated apoptosis pathways, co-staining with organelle markers (such as MitoTracker for mitochondria or nuclear stains) can provide valuable information about DUSP13 subcellular localization and potential translocation during apoptosis .

• Signal amplification considerations: For low-abundance proteins, consider whether direct detection with FITC-conjugated primary antibodies provides sufficient sensitivity or if a detection system using unconjugated primary antibodies with FITC-conjugated secondary antibodies would offer better signal amplification .

• Cross-reactivity assessment: Validate the specificity of your FITC-conjugated DUSP13 antibody using appropriate controls, such as DUSP13 knockout/knockdown cells or peptide competition assays, to ensure the observed staining pattern accurately represents DUSP13 localization .

Careful attention to these technical considerations will enhance the reliability and interpretability of cellular localization studies using FITC-conjugated DUSP13 antibodies.

How can researchers effectively knockdown DUSP13A to study its function in ASK1 signaling?

Effective knockdown of DUSP13A for studying its function in ASK1 signaling requires careful experimental design. Based on published methodologies, the following approach is recommended:

• siRNA design and validation: Small interfering RNA (siRNA) specifically targeting DUSP13A mRNA has been successfully used to knockdown DUSP13A expression. Design siRNAs targeting unique regions of DUSP13A to avoid off-target effects on DUSP13B or other related phosphatases .

• Transfection optimization: For neuroblastoma SK-N-SH cells, which express endogenous DUSP13A, transfection with DUSP13A siRNA expression plasmid has been shown to effectively reduce DUSP13A expression. Optimize transfection conditions (reagent, DNA/siRNA concentration, incubation time) for your specific cell type .

• Knockdown verification: Confirm DUSP13A knockdown at both mRNA level (using qRT-PCR) and protein level (using Western blotting with validated DUSP13A antibodies) .

• Functional assessment of ASK1 activity: After confirming successful DUSP13A knockdown, assess:

  • ASK1 autophosphorylation and kinase activity

  • Phosphorylation status of downstream factors (JNK, p38 MAPK)

  • ASK1-Akt1 interaction (by co-immunoprecipitation)

  • Apoptotic markers (caspase-3/9 activation, cytochrome c release)

  • Cell death rates under various stress conditions

• Rescue experiments: To confirm specificity, perform rescue experiments by re-expressing siRNA-resistant DUSP13A constructs. Compare the effects of wild-type DUSP13A versus the phosphatase-inactive DUSP13A DACS mutant on restoring ASK1 activity .

• Time-course analysis: Consider temporal dynamics by examining ASK1 activity at various time points after DUSP13A knockdown, as compensatory mechanisms may develop over time.

This systematic approach will enable researchers to comprehensively investigate DUSP13A's role in regulating ASK1 signaling pathways and apoptotic processes.

What experimental techniques can be used to assess the interaction between DUSP13A and ASK1?

Multiple complementary techniques can be employed to comprehensively assess the interaction between DUSP13A and ASK1:

• Co-immunoprecipitation (Co-IP): This technique has successfully demonstrated the interaction between full-length ASK1 and DUSP13A in mammalian cells. The protocol involves:

  • Co-transfecting cells with tagged constructs (e.g., FLAG-ASK1 and HA-DUSP13A)

  • Pulling down one protein using tag-specific antibodies (anti-FLAG affinity agarose)

  • Detecting the co-immunoprecipitated partner by immunoblotting (anti-HA antibody)

• Reciprocal Co-IP: To confirm bidirectional interaction, perform the reverse procedure:

  • Immunoprecipitate DUSP13A (e.g., using FLAG-DUSP13A)

  • Detect co-immunoprecipitated ASK1 by immunoblotting

• In vitro binding assays: For direct interaction assessment:

  • Express and purify recombinant DUSP13A (WT or DACS mutant) from E. coli using His-tag purification

  • Incubate with immunoprecipitated ASK1

  • Analyze bound proteins by immunoblotting

• Domain mapping: To identify specific interaction regions:

  • Generate truncated constructs of ASK1 (e.g., N-terminal domain, residues 1-666)

  • Perform Co-IP assays with DUSP13A

  • The N-terminal domain of ASK1 has been identified as the interaction site for DUSP13A

• Competition binding assays: To assess DUSP13A competition with Akt1:

  • Immunoprecipitate ASK1-Akt1 complexes

  • Add increasing amounts of recombinant DUSP13A

  • Analyze changes in Akt1 binding to ASK1

• Functional kinase assays: To assess the impact of interaction on kinase activity:

  • Immunoprecipitate ASK1

  • Add recombinant DUSP13A

  • Measure ASK1 autophosphorylation and substrate (MKK6) phosphorylation

Through these complementary approaches, researchers can thoroughly characterize the physical and functional interaction between DUSP13A and ASK1.

How can the phosphatase activity of DUSP13A be measured in experimental settings?

Measuring the phosphatase activity of DUSP13A requires specific methodologies to accurately assess its enzymatic function:

• Recombinant protein preparation:

  • Express His-tagged DUSP13A proteins in E. coli

  • Purify using Ni-NTA beads (Qiagen)

  • Elute with imidazole

  • Use the purified protein for in vitro assays

• Phosphatase activity assay using synthetic substrate:

  • Reaction conditions: 100 mM Tris-HCl pH 8.2, 40 mM NaCl, 1 mM DTT, 20% glycerol

  • Substrate: 500 μM 3-O-Methylfluorescein Phosphate (OMFP)

  • Incubation temperature: 30°C

  • Detection method: Monitor the production of 3-O-methylfluorescein by measuring:

    • Absorbance change at 490 nm, or

    • Fluorescence change (excitation at 485 nm, emission at 525 nm)

• Comparative analysis with phosphatase-inactive mutant:

  • Generate DUSP13A DACS mutant (catalytically inactive)

  • Perform parallel assays with wild-type and mutant proteins

  • Calculate enzyme kinetic parameters (Km, Vmax)

• Cellular phosphatase activity:

  • Transfect cells with DUSP13A expression constructs

  • Lyse cells and immunoprecipitate DUSP13A

  • Measure phosphatase activity of the immunoprecipitated protein using OMFP or other suitable substrates

  • Include appropriate controls (phosphatase inhibitors, inactive mutants)

• Phospho-specific substrate analysis:

  • While DUSP13A doesn't appear to dephosphorylate pSer-83 of ASK1 directly, its activity against other physiological substrates should be examined

  • Incubate purified DUSP13A with phosphorylated candidate substrates

  • Analyze dephosphorylation using phospho-specific antibodies

This comprehensive approach allows for accurate measurement of DUSP13A phosphatase activity and helps distinguish between its enzymatic function and non-enzymatic regulatory roles in cellular signaling.

What controls should be included when assessing DUSP13A's effect on ASK1-mediated apoptosis?

To rigorously assess DUSP13A's effect on ASK1-mediated apoptosis, researchers should include the following controls:

• Expression controls:

  • Empty vector controls for all transfection experiments

  • Single transfection controls (ASK1 alone, DUSP13A alone)

  • Co-transfection with varying ratios of ASK1:DUSP13A to establish dose-dependency

  • Western blot verification of protein expression levels

• Enzymatic activity controls:

  • Wild-type DUSP13A compared to catalytically inactive DUSP13A DACS mutant

  • Phosphatase activity assays to confirm functional status of both proteins

  • This comparison is crucial as research shows DUSP13A enhances ASK1-mediated apoptosis independently of its phosphatase activity

• Apoptotic pathway verification controls:

  • Cellular fractionation to monitor cytochrome c release from mitochondria to cytosol

  • Immunoblotting for both cleaved and uncleaved forms of caspase-3 and caspase-9

  • Inclusion of organelle purity markers (e.g., COX IV for mitochondria)

  • Caspase activity assays using specific substrates

  • Cell death quantification using multiple methods (e.g., annexin V/PI staining, TUNEL assay)

• Signaling pathway controls:

  • Monitoring phosphorylation status of ASK1 downstream factors (JNK, p38)

  • ASK1 kinase activity assays with MKK6 as substrate

  • Assessment of ASK1 oligomerization state

  • Analysis of ASK1-Akt1 interaction in the presence and absence of DUSP13A

• Specificity controls:

  • siRNA-mediated knockdown of endogenous DUSP13A

  • Rescue experiments with siRNA-resistant DUSP13A constructs

  • Assessment of DUSP13B effects for comparison

• Cell type controls:

  • Comparison between cell lines with different levels of endogenous ASK1 and DUSP13A expression

  • Human neuroblastoma SK-N-SH cells (which express endogenous DUSP13A) versus HEK 293 cells (which do not express endogenous DUSP13A)

This comprehensive set of controls will ensure robust and reproducible assessment of DUSP13A's specific effects on ASK1-mediated apoptotic pathways.