DPP9 Antibody, FITC conjugated

What is the role of DPP9 in cellular processes?

Dipeptidyl peptidase 9 (DPP9) is an aminopeptidase that removes dipeptides from the N-termini of substrates containing proline or alanine at the second position. This enzymatic activity has been implicated in various cellular processes, including cell survival, metabolism, immune regulation, and tumorigenesis . DPP9 influences key pathways such as the N-end rule pathway by modulating protein stability through neo-N-terminal processing. For example, it cleaves Syk kinase to produce a neo N-terminus with serine at position 1, which affects Syk's stability and its subsequent ubiquitination . Additionally, DPP9 plays a role in inhibiting caspase-1-dependent pyroptosis by preventing activation of inflammasomes such as NLRP1 and CARD8 .

How does FITC conjugation enhance the utility of DPP9 antibodies in research?

Fluorescein isothiocyanate (FITC) conjugation provides fluorescent labeling to antibodies, enabling visualization and quantification of target proteins in various experimental setups. FITC-conjugated DPP9 antibodies are particularly useful for immunofluorescence microscopy, flow cytometry, and proximity ligation assays (PLA) . These techniques allow researchers to study the spatial distribution of DPP9 within cells and its interactions with other proteins such as Filamin A (FLNA) and Syk kinase . The fluorescence emitted by FITC facilitates high-resolution imaging and real-time tracking of molecular interactions.

What experimental methods can be used to study DPP9 interactions in cells?

Several advanced techniques are available for studying DPP9 interactions:

• Co-immunoprecipitation (Co-IP): This method isolates protein complexes involving DPP9 by using specific antibodies against DPP9 or its interacting partners like FLNA .

• Proximity ligation assays (PLA): PLA enables visualization of protein-protein interactions at single-molecule resolution within cells. For example, PLA has been used to detect interactions between DPP9 and FLNA in HeLa cells .

• Western blotting: This technique quantifies DPP9 expression levels and assesses post-translational modifications under different experimental conditions .

• Immunofluorescence microscopy: FITC-conjugated antibodies facilitate the localization of DPP9 within cellular compartments .

• Gene silencing: RNA interference (e.g., shRNA) can be employed to knock down DPP9 expression and investigate its functional consequences on cell proliferation, migration, and signaling pathways .

How does DPP9 regulate immune responses?

DPP9 acts as a critical regulator of immune responses by inhibiting inflammasome activation. It prevents oligomerization and activation of NLRP1 and CARD8 inflammasomes through its dipeptidyl peptidase activity . Although neither NLRP1 nor CARD8 are direct substrates of DPP9, the enzyme sequesters cleaved fragments of these proteins in a ternary complex, thereby suppressing pyroptosis in monocytes and macrophages . This regulatory mechanism underscores the importance of DPP9 in maintaining immune homeostasis.

What are the implications of DPP9 inhibition in cancer research?

Inhibition of DPP9 has significant implications for cancer research:

How can researchers address data contradictions when studying DPP9?

Addressing data contradictions requires methodological rigor:

• Replication Studies: Perform experiments under identical conditions to verify initial findings.

• Use of Multiple Assays: Employ diverse techniques such as Co-IP, PLA, Western blotting, and immunofluorescence to cross-validate results.

• Quantitative Analysis: Utilize statistical tools to analyze data from independent experiments.

• Control Experiments: Include appropriate positive and negative controls to rule out artifacts.

• Review Literature: Compare findings with published studies to identify potential discrepancies or confirmatory evidence.

For example, discrepancies regarding the role of DPP9 in inflammasome regulation may arise due to differences in experimental models or assay sensitivity .

What factors influence the specificity of FITC-conjugated antibodies?

The specificity of FITC-conjugated antibodies depends on several factors:

• Antibody Affinity: High-affinity antibodies ensure precise binding to target epitopes.

• Conjugation Efficiency: Optimal labeling with FITC minimizes non-specific fluorescence.

• Experimental Conditions: Proper pH and buffer composition prevent antibody degradation during assays.

• Cross-reactivity Testing: Validate antibody specificity against closely related proteins such as DPP8 or DPPIV .

Ensuring specificity is critical for accurate interpretation of experimental data.

How can researchers optimize experimental design when using FITC-conjugated DPP9 antibodies?

Optimizing experimental design involves careful planning:

• Selection of Controls: Include unstained samples and isotype controls to account for background fluorescence.

• Antibody Dilution: Determine optimal dilution ratios through titration experiments.

• Fluorescence Detection Settings: Adjust excitation/emission wavelengths on imaging platforms to match FITC properties.

• Temporal Studies: Conduct time-course experiments to monitor dynamic changes in protein localization or interaction.

For example, immunofluorescence studies using FITC-conjugated antibodies require precise calibration to differentiate specific signals from autofluorescence .

What challenges exist in studying protein-protein interactions involving DPP9?

Challenges include:

• Transient Interactions: Many protein-protein interactions involving DPP9 are transient and require stabilization using cross-linkers during Co-IP assays .

• Low Abundance Proteins: Detecting low-abundance proteins necessitates highly sensitive detection methods like PLA.

• Complex Pathways: Interactions between DPP9, FLNA, Syk kinase, and other partners involve intricate signaling pathways that require comprehensive analysis using multiple techniques .

Overcoming these challenges demands methodological innovation and technical expertise.

How does substrate specificity affect the function of DPP9?

DPP9 exhibits strict substrate specificity by cleaving N-terminal dipeptides from proteins containing proline or alanine at position 2. This specificity influences its role in regulating protein stability and signaling pathways. For instance:

• Neo-N-terminal Processing: Cleavage by DPP9 generates neo-N-terminal residues that determine substrate susceptibility to ubiquitination or degradation .

• Inflammasome Regulation: Substrate specificity is essential for inhibiting NLRP1 and CARD8 inflammasomes despite these proteins not being direct substrates .

Understanding substrate specificity provides insights into the molecular mechanisms underlying DPP9's functions.