DPP9 Antibody

What is DPP9 and why is it significant in cellular pathways?

DPP9 (Dipeptidyl Peptidase 9) is an aminopeptidase that specifically removes dipeptides from N-termini of substrates having a proline or alanine in second position. It plays crucial roles in several cellular pathways including cell survival, metabolism, and immune signaling. Notably, DPP9 has been identified as a novel component of the N-end rule pathway, which governs protein degradation based on N-terminal amino acids . This makes DPP9 a significant target for research in areas ranging from basic cell biology to cancer and immunology. Studies have shown that mice born with inactive DPP9 die shortly after birth, indicating its essential role in development . DPP9 has also been linked to regulation of NQO1 and intracellular ROS, potentially contributing to chemotherapy resistance in liver cancer . Its diverse functions make it relevant to multiple research fields and potential therapeutic applications.

What detection methods are most effective for DPP9 in different experimental contexts?

Multiple validated detection methods can be employed for DPP9 analysis, each with specific advantages:

• Western Blot: Effective for detecting DPP9 protein levels in cell lysates or tissue samples. Anti-human DPP9 monoclonal antibodies can detect DPP9 at approximately 100-104 kDa under reducing conditions . This method is particularly valuable for quantitative analysis of total DPP9 expression.

• Immunofluorescence: Useful for visualizing DPP9 cellular localization. Studies have shown DPP9 localization in both cytoplasm and nuclei of cells like HeLa . Standard protocols typically use 8 μg/mL of Anti-Human DPP9 Monoclonal Antibody with NorthernLights™ 557-conjugated secondary antibody and DAPI counterstaining.

• Proximity Ligation Assay (PLA): Particularly valuable for detecting protein-protein interactions involving DPP9, such as its interaction with Filamin A (FLNA). Each PLA dot represents a single DPP9-protein interaction event . This technique has successfully demonstrated DPP9's interactions in situ.

• Activity Assays: Using artificial substrates like GP-AMC to measure DPP9 enzymatic activity in cell lysates, allowing researchers to assess functional activity rather than just protein presence . This approach is essential for determining if detected DPP9 is enzymatically active.

The choice of method depends on whether you're investigating protein levels, localization, interactions, or enzymatic activity of DPP9.

How can I distinguish between DPP9 and other DPP family members in my experiments?

Distinguishing between DPP9 and related family members (DPP4, DPP8) requires careful experimental design:

• Antibody Selection: Use validated antibodies with confirmed specificity for DPP9. For example, Mouse Anti-Human DPP9 Monoclonal Antibody (Clone #988515) has been validated for specific detection of DPP9 . Verify specificity through knockdown controls.

• Molecular Weight Differentiation: DPP9 appears at approximately 100-104 kDa on Western blots , which can help distinguish it from other DPP family members with different molecular weights.

• Selective Inhibitors: While complete selectivity remains challenging, inhibitors like 1G244 target DPP8 and DPP9 but not DPP4 . Comparing effects of selective versus non-selective inhibitors can help differentiate between family members.

• siRNA/shRNA Knockdown: Targeted silencing of DPP9 can confirm specificity of signals in detection methods . Control experiments should include knockdowns of other DPP family members to rule out cross-reactivity.

• Immunofluorescence Patterns: DPP family members may have distinct subcellular localization patterns that can be visualized through co-staining experiments . For example, immunofluorescence images of DG-75 cells have shown distinct localization patterns for DPP9 compared to DPP8 and DPPIV.

A combination of these approaches provides the most reliable differentiation between DPP9 and other family members.

What controls should be included when using DPP9 antibodies for Western blot analysis?

Robust Western blot experiments with DPP9 antibodies require several critical controls:

• Positive Control: Include lysates from cells known to express DPP9 at detectable levels, such as HeLa cells, K562 human chronic myelogenous leukemia cell line, or human heart tissue, which have been confirmed to express DPP9 in previous studies .

• Negative Control: Use lysates from cells with DPP9 knockdown (siRNA or shRNA) to demonstrate antibody specificity . The significant reduction in band intensity validates that the observed band is indeed DPP9.

• Loading Control: Include antibodies against housekeeping proteins like tubulin (as demonstrated in DPP9 expression studies in DG-75 cells) to normalize protein loading across samples .

• Molecular Weight Marker: Always include a protein standard to confirm that the detected band appears at the expected molecular weight for DPP9 (approximately 100-104 kDa) .

• Cell Treatment Controls: When studying DPP9 in specific signaling contexts, include appropriate cell stimulation controls. For instance, DG-75 cells stimulated with F(ab')2 fragment goat-anti-human IgG+IgM can serve as controls for B-cell activation studies .

• Cross-Reactivity Control: Test the antibody on samples from different species if working with non-human models to ensure the antibody recognizes the target across species of interest.

These controls collectively ensure reliable and interpretable results when using DPP9 antibodies for Western blot analysis.

How should I optimize immunofluorescence protocols for DPP9 detection in different cell types?

Optimizing immunofluorescence protocols for DPP9 detection requires consideration of several factors:

• Fixation Method:

  • Paraformaldehyde (4%) is commonly used but may mask certain epitopes

  • Immersion fixation has been successfully used for DPP9 detection in HeLa cells

  • Test different fixation protocols to determine which best preserves DPP9 epitopes in your cell type

• Antibody Concentration:

  • Titrate primary antibody concentration (typically starting at 1-10 μg/mL)

  • Published studies have used 8 μg/mL of Anti-Human DPP9 Monoclonal Antibody for HeLa cells

  • Optimize secondary antibody dilutions to maximize signal while minimizing background

• Incubation Conditions:

  • Primary antibody incubation times vary (published protocols suggest 3 hours at room temperature for DPP9 staining in HeLa cells)

  • Temperature affects binding kinetics; compare room temperature versus 4°C incubation

• Counterstaining:

  • Use DAPI for nuclear counterstaining to better visualize the nuclear component of DPP9 staining

  • Consider co-staining with markers for cellular compartments to confirm DPP9 localization

• Controls:

  • Include cells with DPP9 knockdown as negative controls to confirm antibody specificity

  • Use secondary-only controls to assess non-specific binding

  • Consider co-staining for other DPP family members to establish specificity

Following these optimization steps enables reliable detection of DPP9 across different cell types while ensuring specificity of the observed signals.

What are the key considerations for designing DPP9 knockdown or overexpression experiments?

Designing effective DPP9 knockdown or overexpression experiments requires careful planning:

• Knockdown Approaches:

  • siRNA/shRNA: Research has successfully used these approaches for transient and stable DPP9 knockdown in multiple cell types

  • CasRx-based systems: Have been successfully used for DPP9 knockdown in neurological studies

  • Verify knockdown efficiency by both Western blot and functional activity assays to ensure both protein level and enzymatic activity are reduced

• Overexpression Systems:

  • Research has successfully used DPP9 overexpression vectors delivered via AAV in hippocampal studies

  • Consider adding tags for easier detection, but verify they don't interfere with DPP9 function

  • Ensure proper subcellular localization of overexpressed DPP9 matches endogenous patterns

• Delivery Methods:

  • Transfection efficiency varies by cell type; optimize protocols accordingly

  • AAV-based delivery has been successfully used for hippocampal DPP9 manipulation

  • For in vivo studies, expression typically stabilizes after 21 days with AAV delivery

• Validation:

  • Confirm knockdown/overexpression at both mRNA (qPCR) and protein (Western blot) levels

  • Assess enzymatic activity using DPP9 substrates to confirm functional alterations

  • Western blot analysis has been successfully used to confirm DPP9 knockdown in multiple cell lines

• Functional Readouts:

  • Include assays relevant to known DPP9 functions (cell survival, protein degradation, etc.)

  • For immune cells, monitor Syk stability and signaling

  • For neurological studies, electrophysiological measurements like LTP have proven informative

  • For cancer studies, assess effects on NQO1 expression and ROS levels

These considerations help ensure robust and interpretable results in DPP9 manipulation experiments.

How can DPP9 antibodies be used to investigate the interaction between DPP9 and Filamin A?

Investigating the DPP9-Filamin A interaction requires specialized approaches with DPP9 antibodies:

• Co-immunoprecipitation (Co-IP):

  • Use DPP9 antibodies to pull down protein complexes from cell lysates

  • To stabilize transient interactions, treat cells with cross-linkers like DPDPB (containing a spacer of 19.9 Å), which has been shown to effectively preserve the DPP9-FLNA interaction

  • Western blot for Filamin A in the precipitated material to confirm the interaction

• Proximity Ligation Assay (PLA):

  • This technique allows visualization of protein interactions in situ

  • Use primary antibodies against DPP9 and Filamin A

  • Each PLA dot represents a single interaction event

  • This method has successfully demonstrated DPP9-FLNA interactions in previous studies, with significantly reduced signals in FLNA-silenced control cells

• Immunofluorescence Co-localization:

  • Perform double immunofluorescence staining with DPP9 and Filamin A antibodies

  • Research has shown an overlap in the cellular localization of these two proteins in HeLa cells

  • This approach provides spatial information about where in the cell the interaction occurs

• Functional Validation:

  • Research has shown that FLNA repeat 5 is required for the interaction with DPP9

  • This interaction recruits DPP9 to Syk, indicating that FLNA serves as a scaffold connecting these proteins

  • Monitor downstream effects on Syk processing and stability to confirm functional relevance

These approaches, centered around DPP9 antibodies, provide complementary information about the nature, location, and function of the DPP9-Filamin A interaction.

What methodologies can be used to study DPP9's role in the N-end rule pathway and protein degradation?

Studying DPP9's role in the N-end rule pathway requires sophisticated methodologies:

• N-terminal Sequencing:

  • Use mass spectrometry to identify N-terminal amino acids of potential DPP9 substrates

  • Compare N-termini in DPP9-inhibited versus normal conditions

  • This approach has confirmed DPP9's cleavage activity on Syk, identifying the neo-N-terminus with serine at position 1

• Pulse-Chase Experiments:

  • Label proteins with radioactive amino acids (pulse) and track their degradation over time (chase)

  • Compare protein half-lives in cells with normal versus inhibited/knocked down DPP9

  • This method has been effectively used to demonstrate that DPP9 processing influences Syk stability

• Ubiquitination Assays:

  • Immunoprecipitate substrate proteins (e.g., Syk) and blot for ubiquitin

  • DPP9 silencing has been shown to reduce Cbl (E3 ligase) interaction with Syk, suggesting DPP9 processing is a prerequisite for ubiquitination

  • This approach connects DPP9 activity to the ubiquitin-proteasome system

• Mutagenesis Studies:

  • Generate mutants of potential substrates with altered N-terminal sequences

  • Assess how mutations affect processing by DPP9 and subsequent protein stability

  • Research has shown that mutations at the Ser1 position of Syk strongly influence its stability

• Inhibitor Studies:

  • Use DPP9 inhibitors (e.g., 1G244) to block enzymatic activity

  • Compare substrate stability and degradation with and without inhibition

  • Research has shown that DPP9 inhibition stabilizes Syk, modulating signaling pathways

These methodologies provide complementary approaches to understanding DPP9's role in the N-end rule pathway and protein degradation.

How can I investigate DPP9's function in B-cell signaling and Syk regulation?

Investigating DPP9's role in B-cell signaling and Syk regulation requires specialized techniques:

• B-cell Activation Models:

  • Use established B-cell activation protocols, such as stimulation with F(ab')2 fragment goat-anti-human IgG+IgM (12 μg/ml)

  • Monitor DPP9 levels and localization before and after activation

  • Studies in DG-75 cells have successfully used this approach to investigate DPP9 in B-cell contexts

• DPP9-Syk Interaction Studies:

  • Use Proximity Ligation Assay (PLA) to visualize DPP9-Syk interactions in situ

  • Determine if the interaction is direct or mediated by Filamin A

  • Research has shown that Filamin A recruits DPP9 to Syk, establishing a link between these proteins

• Syk Processing Analysis:

  • Use N-terminal antibodies or mass spectrometry to detect DPP9-mediated cleavage of Syk

  • Research has shown that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1

  • This processing is critical for subsequent Syk degradation

• Syk Stability Assessment:

  • Perform pulse-chase experiments to track Syk half-life with and without DPP9 activity

  • Research has shown that DPP9 preferentially cleaves the active form of Syk, acting as a shut-off mechanism

  • DPP9 inhibition stabilizes Syk, thereby modulating Syk signaling

• Syk Ubiquitination:

  • Immunoprecipitate Syk and blot for ubiquitin under various DPP9 conditions

  • Monitor Cbl (E3 ligase) recruitment to Syk with and without DPP9

  • DPP9 silencing reduces Cbl interaction with Syk, suggesting DPP9 processing is a prerequisite for ubiquitination

These approaches provide a comprehensive framework for investigating DPP9's function in B-cell signaling and Syk regulation, revealing its role as a negative regulator of this important signaling pathway.

How can researchers investigate DPP9's role in cancer biology and therapeutic resistance?

DPP9's emerging role in cancer biology can be investigated through several approaches:

• Expression Analysis in Cancer Models:

  • Western blot analysis shows variable DPP9 expression across cancer cell lines

  • Liver cancer studies have shown DPP9 involvement in chemotherapy resistance

  • Compare expression between sensitive and resistant cell populations

• Mechanisms of Treatment Resistance:

  • Research has shown that DPP9 weakens responses of liver cancer cells to chemotherapy drugs by up-regulating NQO1 and inhibiting intracellular ROS

  • Quantify NQO1 mRNA and protein levels after DPP9 overexpression or silencing

  • Western blot assays have demonstrated that NQO1 protein levels in cells with DPP9 overexpression and silencing are significantly up-regulated and down-regulated, respectively

• Cell Viability Studies:

  • Multiple cancer cell lines (MM.1S, KARPAS299, THP-1, KG1, Daudi, NAMALWA) show differential sensitivity to DPP9 inhibitors

  • Use colorimetric assays (WST-1) to assess cell number after treatment with DPP9 inhibitors at various concentrations and timepoints

  • Complement with LDH release assays to measure cytotoxicity

• In Vivo Tumor Models:

  • Studies have shown that DPP8 selective inhibitor tominostat has antitumor effects in mouse models

  • Establish xenograft models using sensitive cell lines (e.g., MM.1S)

  • Monitor tumor volume after DPP9 inhibitor administration

These approaches provide a framework for investigating DPP9's complex roles in cancer biology, potentially leading to novel therapeutic strategies targeting chemotherapy resistance mechanisms.

What approaches can be used to study DPP9's role in neurological function?

Investigating DPP9's neurological functions requires specialized techniques:

These techniques collectively provide robust evidence for DPP9's bidirectional regulation of hippocampal function, particularly in synaptic plasticity mechanisms like LTP.

How can I assess the therapeutic potential of DPP9 inhibitors?

Evaluating DPP9 inhibitors for therapeutic applications requires systematic investigation:

• Cell Line Screening:

  • Test multiple cancer cell lines with various DPP9 inhibitors (1G244, talabostat, tominostat)

  • Research has shown differential sensitivity across cell lines (MM.1S and KARPAS299 are sensitive, while Daudi cells are resistant)

  • Include both short-term (6h) and long-term (72h) treatments to distinguish between acute and sustained effects

• Mechanism of Action Studies:

  • Determine if cytotoxicity occurs through pyroptosis or other cell death mechanisms

  • Research has linked DPP9 inhibitor sensitivity to expression of specific proteins including HCK, CARD8, caspase-1, and GSDMD

  • Western blot analysis can be used to assess expression of these key proteins in cell lines with different sensitivities

• Validation with Genetic Approaches:

  • Compare effects of pharmacological inhibition with DPP9 knockdown

  • Studies have used knockdown of DPP8 and DPP9 in MM.1S or KARPAS299 cells to confirm specificity of inhibitor effects

  • This approach helps distinguish between on-target and off-target effects

• In Vivo Efficacy and Safety:

  • Subcutaneous inoculation of cancer cells (e.g., MM.1S or Daudi) into mouse models

  • Administer DPP9 inhibitors and monitor tumor volume over time

  • Studies have shown that tominostat has potent antitumor effects in vivo

  • Monitor body weight and other parameters to assess potential side effects

• Application in Specific Disease Contexts:

  • For liver cancer, investigate DPP9 inhibition as a strategy to overcome chemotherapy resistance

  • For neurological applications, consider how DPP9 inhibition affects synaptic plasticity and learning

  • For immune-related diseases, focus on effects on B-cell signaling and Syk regulation

These approaches provide a comprehensive framework for evaluating the therapeutic potential of DPP9 inhibitors across multiple disease contexts.

How can I address variability in DPP9 antibody performance across different applications?

Addressing variability in DPP9 antibody performance requires systematic optimization:

• Antibody Selection:

  • Different antibodies may perform differently across applications

  • Monoclonal antibodies like Clone #988515 have been validated for multiple applications including Western blot, immunofluorescence, and Simple Western™

  • Confirm that your antibody has been validated for your specific application

• Epitope Accessibility:

  • Different experimental conditions may affect epitope exposure

  • For Western blotting, ensure complete protein denaturation using appropriate reducing conditions and Immunoblot Buffer Group 1

  • For immunofluorescence, optimize fixation methods to preserve epitope accessibility

• Cross-Reactivity Assessment:

  • Test antibody specificity using DPP9 knockdown controls

  • Western blot analysis following DPP9 silencing can confirm antibody specificity

  • This approach helps distinguish between specific and non-specific signals

• Protocol Optimization:

  • For Western blot, test different protein loading amounts (studies have used 10 μg per lane)

  • For immunofluorescence, optimize antibody concentration (8 μg/mL has been effective for HeLa cells)

  • For activity assays, ensure appropriate substrate concentration (250 μM GP-AMC has been used successfully)

• Validation Across Methods:

  • Confirm findings using complementary detection methods

  • Correlate protein detection with functional activity measurements

  • For example, complement Western blot protein detection with enzymatic activity assays using GP-AMC substrate

These approaches help ensure reliable and reproducible results when working with DPP9 antibodies across different experimental contexts.

What are the critical variables to control when using DPP9 inhibitors in experimental settings?

Effective use of DPP9 inhibitors requires control of several critical variables:

• Inhibitor Specificity:

  • Most available inhibitors target both DPP8 and DPP9

  • 1G244 inhibits both DPP8/9 but not unrelated substrates like R-AMC

  • Complement inhibitor studies with genetic approaches (knockdown/overexpression) to confirm specificity

• Dose-Response Relationships:

  • Inhibitor effects are highly dose-dependent

  • Studies typically test a range of concentrations (0-100 μM)

  • Different cell types may require different inhibitor concentrations for equivalent target engagement

• Treatment Duration:

  • Short-term (6h) versus long-term (72h) treatments may yield different results

  • Cytotoxicity is often assessed after 6h using LDH release assays

  • Cell viability over longer periods (72h) may be measured using WST-1 assays

• Cell Type Considerations:

  • Significant variability exists in sensitivity across cell types

  • MM.1S and KARPAS299 cells show high sensitivity, while Daudi cells are resistant

  • These differences correlate with expression of proteins like HCK, CARD8, and GSDMD

• Vehicle Controls:

  • Include appropriate DMSO controls at equivalent concentrations

  • Control for potential vehicle effects on cell function and viability

• Validation of Inhibition:

  • Confirm reduced DPP activity using enzymatic assays

  • Studies have successfully used the artificial DPP substrate GP-AMC (250 μM) to confirm inhibition

  • Compare with unrelated substrates (e.g., R-AMC) to confirm specificity

Controlling these variables ensures reliable and reproducible results when using DPP9 inhibitors in experimental settings.

How can apparent contradictions in DPP9 research findings be reconciled?

Reconciling contradictory findings in DPP9 research requires careful analysis of experimental contexts:

• Cell Type Considerations:

  • DPP9 functions appear to be highly cell type-specific

  • For example, DPP9 regulates NQO1 in liver cancer cells but interacts with NPY in hippocampal neurons

  • These distinct interactions likely reflect different molecular environments across cell types

• Experimental Timeframes:

  • Acute versus chronic DPP9 inhibition or knockdown may yield different results

  • Short-term (6h) versus long-term (72h) inhibitor treatments show different response profiles

  • Consider temporal aspects when comparing studies

• Expression Level Differences:

  • Baseline DPP9 expression varies significantly across cell types

  • Western blot analysis shows variable expression across cancer cell lines and tissues

  • Higher expression levels may require higher inhibitor concentrations for complete suppression

• Methodological Variations:

  • Different detection methods have different sensitivities and limitations

  • Comparing studies using different methodologies requires careful consideration of these differences

  • Standardize experimental approaches when directly comparing results

• Functional Context:

  • DPP9's role as both an enzyme and a scaffold protein may lead to apparently contradictory findings

  • Some effects may depend on enzymatic activity while others rely on protein-protein interactions

  • Distinguish between these functions through careful experimental design

• Activation State Dependencies:

  • DPP9 preferentially cleaves the active form of Syk , suggesting its function may depend on cellular activation state

  • Consider activation status when comparing results across studies

By systematically analyzing these factors, researchers can reconcile apparently contradictory findings and develop a more comprehensive understanding of DPP9's complex biological roles.