DUSP10 Antibody

What is DUSP10 and what are its primary cellular functions?

DUSP10 is a protein phosphatase involved in the inactivation of MAP kinases, with specificity for the MAPK11/MAPK12/MAPK13/MAPK14 subfamily. It preferentially dephosphorylates and inactivates p38α MAP kinase and JNK, but does not appear to affect p44/42 MAPK . DUSP10 plays significant roles in both innate and adaptive immune responses, inflammation regulation, and adipocyte differentiation. The protein's expression can be either constitutive or inducible depending on the cell type - constitutively expressed in naïve mouse lymphocytes but downregulated following T-cell receptor activation, while not constitutively expressed in macrophages but upregulated after LPS stimulation .

What is the expected molecular weight for detecting DUSP10 via Western blotting?

The expected molecular weight for DUSP10 detection in Western blotting is approximately 53-54 kDa. According to the product information from Cell Signaling Technology, the molecular weight (MW) of DUSP10 is 54 kDa . This is consistent with the predicted band size of 53 kDa reported by Abcam for their anti-DUSP10 antibody (ab228987) . When running SDS-PAGE for DUSP10 detection, a 10% gel is typically suitable, as demonstrated in the validation experiments for the ab228987 antibody .

What are the optimal dilutions and applications for DUSP10 antibodies?

The optimal applications and dilutions for DUSP10 antibodies vary based on the specific antibody and experimental design. Based on the provided search results:

Always validate these dilutions for your specific experimental conditions, as optimal concentrations may vary depending on sample type, antibody lot, and detection method. For Western blotting, ensure proper optimization with positive controls and loading controls for accurate interpretation.

How can I validate DUSP10 antibody specificity for my research?

Validating antibody specificity is crucial for ensuring reliable research results. For DUSP10 antibodies, consider implementing these methodological approaches:

• Positive and negative controls: Use cell lines known to express DUSP10 (e.g., HeLa, HCT116) as positive controls. For negative controls, consider using DUSP10 knockout cells or siRNA-mediated knockdown samples.

• Catalytically inactive mutant controls: Utilize the catalytically inactive DUSP10-CS mutant (where Cys-408 is replaced by Ser) as described in the brown adipocyte differentiation research . This approach allows you to distinguish between phosphatase activity-dependent effects and other protein interactions.

• Overexpression validation: Create DUSP10 overexpression models using retroviral systems (e.g., pRetroX-IRES-ZsGreen1 vector) to confirm antibody specificity against elevated protein levels .

• Knockdown validation: Implement shRNA approaches using vectors like pSIREN-RetroQ-DsRed Express with targeted sequences to verify antibody signal reduction correlates with protein reduction .

• Multiple detection methods: Confirm DUSP10 expression across different techniques (Western blot, IHC, ICC/IF) to ensure consistent results.

How does DUSP10 expression affect p38 MAPK signaling pathway analysis?

DUSP10 selectively dephosphorylates and inactivates p38α MAP kinase, making it a critical regulator of p38 MAPK signaling. When designing experiments to study this pathway:

• Temporal dynamics: DUSP10 overexpression induces reduction in active p38 levels, particularly in early stages of cellular differentiation processes. Research shows this effect is most pronounced in early stages but continues through later stages (up to 6 days) in brown adipocyte differentiation .

• Phosphorylation monitoring: Track phosphorylated p38 (p-p38) levels via Western blotting when manipulating DUSP10 expression. In overexpression systems, p-p38 is consistently downregulated until late stages of differentiation .

• Pathway specificity: While DUSP10 significantly impacts p38 signaling, it shows selective effects on MAP kinases. For instance, studies demonstrate that p-JNK and JNK levels show no significant expression changes upon DUSP10 ectopic expression in certain cell types .

• Functional validation: Confirm the phosphatase activity's importance by comparing wild-type DUSP10 against catalytically inactive DUSP10-CS mutant. The mutant demonstrates no significant changes in p38 phosphorylation, confirming that phosphatase activity is critical for the observed effects .

What immunological parameters should be analyzed when studying DUSP10 in immune contexts?

DUSP10 plays important roles in both innate and adaptive immune responses. When investigating DUSP10 in immunological research:

• Cytokine profiling: DUSP10 acts as a negative regulator of both Type 1 (IFN-γ) and Type 2 (TNF-α) cytokine expression in effector CD4 and CD8 T-cells. Monitor these cytokines to assess DUSP10's impact on immune responses .

• T-cell activation markers: Since DUSP10 reduces AP-1 expression through regulating JNK activity, measure AP-1 levels alongside T-cell activation markers .

• Antigen presentation capacity: CD11c+ dendritic cells from DUSP10-deficient mice show enhanced antigen presentation activity to splenic CD4+ T-cells, resulting in increased IFN-γ production. Assess dendritic cell function when manipulating DUSP10 levels .

• Immune cell infiltration analysis: Use single-sample Gene Set Enrichment Analysis (ssGSEA) to evaluate the abundance of immune-connected indicators and assess correlation between DUSP10 expression and immune infiltration .

• Immune score correlation: Research shows DUSP10 expression positively correlates with ESTIMATE, stromal, and immune scores while inversely linking to tumor purity in certain cancers .

How can DUSP10 be effectively studied in the context of cancer research?

DUSP10 has emerging roles in cancer biology, particularly in lower-grade glioma (LGG). When investigating DUSP10 in cancer contexts:

• Expression profiling: Perform pan-cancer analysis to determine expression patterns across tumor types. This approach has revealed specific roles for DUSP10 in certain cancers like LGG .

• Clinicopathologic correlation: Thoroughly examine the relationship between DUSP10 expression and clinicopathologic features, prognosis, biological processes, immune traits, gene variations, and treatment responses .

• Immune microenvironment assessment: Implement the CIBERSORT algorithm to investigate infiltration abundances of tumor-infiltrating immune cells (TIICs) between high and low DUSP10-expressing tumors .

• Functional enrichment analysis: Conduct GO-BP and KEGG analyses to elucidate the biological pathways associated with DUSP10 expression in cancer contexts. Research has shown connections between DUSP10 and immune regulation in LGG specifically .

• In vitro functional studies: Design experiments to detect the underlying functions of DUSP10 in cancer cell lines, as performed in LGG research .

What are common issues when using DUSP10 antibodies in Western blotting?

When working with DUSP10 antibodies in Western blotting applications, researchers may encounter several challenges:

• Band specificity: DUSP10 antibodies should detect a band at approximately 53-54 kDa. If multiple bands appear, optimization of antibody dilution (typically 1:1000) and blocking conditions may be necessary .

• Sample preparation: Ensure proper cell lysis and protein extraction methods that preserve phosphatase activity. Standard lysis buffers containing phosphatase inhibitors may be required to prevent post-lysis dephosphorylation events.

• Loading controls: Due to DUSP10's role in signaling pathways, expression levels may vary significantly between samples. Use appropriate loading controls and consider normalizing to total protein rather than housekeeping genes when comparing expression across different conditions.

• Detection sensitivity: For endogenous DUSP10 detection, enhanced chemiluminescence (ECL) technique has been successfully employed . If signal strength is insufficient, consider using more sensitive detection systems or increasing protein loading (30 μg of total protein per lane has been validated for HeLa and HCT 116 whole cell lysates) .

What considerations are important for immunohistochemistry and immunofluorescence applications with DUSP10 antibodies?

When utilizing DUSP10 antibodies for immunohistochemistry (IHC) or immunofluorescence (IF) applications:

• Fixation methods: For immunofluorescence, paraformaldehyde fixation has been validated for DUSP10 detection in HeLa cells . For IHC, standard paraffin embedding and antigen retrieval protocols have been successful with DUSP10 antibodies at 1:100 dilution .

• Signal localization: Confirm subcellular localization patterns align with DUSP10's known distribution. Nuclear counterstaining (e.g., with Hoechst 33342) can help contextualize DUSP10 staining patterns .

• Positive controls: Use validated cell lines like HeLa for immunofluorescence or known DUSP10-expressing tissues like U87 xenograft tissue for IHC validation .

• Background reduction: Optimize blocking conditions to minimize non-specific binding, particularly in tissues with high endogenous phosphatase activity. Consider using specialized blocking reagents for phosphatase detection.

• Dual staining approaches: When studying DUSP10's interaction with signaling partners like p38 or JNK, consider dual immunofluorescence to simultaneously visualize both proteins and assess co-localization patterns.

What cellular and animal models are most appropriate for studying DUSP10 function?

Selecting appropriate models for DUSP10 research depends on your specific research questions:

• Cell line models:

  • HeLa and HCT 116 cell lines have been validated for DUSP10 expression studies via Western blotting

  • For immunofluorescence, HeLa cells have demonstrated clear DUSP10 staining patterns

  • Primary brown preadipocytes have been used successfully to study DUSP10's role in adipocyte differentiation

• Animal models:

  • DUSP10-deficient mice have been instrumental in understanding this phosphatase's role in immune responses

  • These knockout models demonstrate enhanced antigen presentation by dendritic cells to CD4+ T-cells

  • DUSP10-deficient mice show resistance to experimental myelin-induced autoimmune encephalitis (EAE), an animal model of multiple sclerosis

• Genetic modification approaches:

  • Retrovirus-mediated infection systems using pRetroX-IRES-ZsGreen1 vector have successfully established stable DUSP10 expression

  • For knockdown studies, shRNA approaches using pSIREN-RetroQ-DsRed Express retroviral vector with specific DUSP10-targeting sequences have achieved approximately 50% reduction in expression

  • Catalytically inactive DUSP10 mutants (Cys-408 to Ser) serve as excellent functional controls

How should expression systems be designed for studying DUSP10 phosphatase activity?

When designing expression systems to study DUSP10 phosphatase activity:

• Vector selection: Retroviral vectors like pRetroX-IRES-ZsGreen1 have been successfully used for stable DUSP10 expression . This system allows for fluorescent tracking (via ZsGreen1) of transduced cells.

• Transfection methodology: For virus production, co-transfection of packaging cells (e.g., GP2-293) with the retroviral vector and VSV-G plasmid using Lipofectamine 2000 has been effective .

• Cell selection: Fluorescence-activated cell sorting (FACS) enables enrichment of infected cells. Selection markers like GFP allow verification of expression through fluorescence microscopy .

• Functional controls: Generate and include the catalytically inactive DUSP10-CS mutant (Cys-408 to Ser) as a critical control to distinguish between phosphatase activity-dependent effects and other protein interactions .

• Expression validation: Confirm DUSP10 expression by Western blot analysis and verify functional activity by assessing downstream target phosphorylation status (particularly p-p38 levels) .

• Inducible systems: For temporal control of DUSP10 expression, consider tetracycline-inducible systems, particularly when studying processes where timing of phosphatase activity is critical.