ddx-15 Antibody

What is DDX15/DHX15 and what are its primary biological functions?

DDX15, also designated as DHX15, DBP1, or HRH2, is a nuclear ATP-dependent RNA helicase belonging to the DEAH-box subfamily of DEAD-box proteins. It is characterized by the conserved motif Asp-Glu-Ala-Asp and functions as a putative RNA helicase . DDX15 is 795 amino acids in length with a calculated molecular weight of 91 kDa, though it is typically observed at 90-95 kDa in experimental contexts .

DDX15 serves dual critical functions in cellular biology:

• Pre-mRNA Processing: DDX15 functions as a pre-mRNA processing factor involved in spliceosome disassembly after mature mRNA release. In cooperation with TFIP11, it facilitates the transition of the U2, U5, and U6 snRNP-containing IL complex to the snRNP-free IS complex, promoting efficient debranching and turnover of excised introns .

• Antiviral Innate Immunity: DDX15 plays a key role in antiviral defense through multiple mechanisms:

  • Acts as an RNA virus sensor by recognizing and binding viral double-stranded RNA (dsRNA)

  • Activates MAVS-dependent signaling to produce interferon-beta and interferon lambda-3

  • Functions in conjunction with NLRP6 to activate inflammasome responses in intestinal epithelial cells

  • Associates with RIG-I caspase activation and recruitment domains (CARDs) to enhance innate immune signaling

Additionally, DDX15 has been implicated in antibacterial innate immunity by promoting Wnt-induced antimicrobial protein expression in Paneth cells .

What types of DDX15 antibodies are available for research applications?

Researchers have access to several types of DDX15 antibodies with varying characteristics:

Each antibody offers distinct advantages depending on experimental needs, such as species reactivity, application compatibility, and epitope specificity. The availability of both monoclonal and polyclonal options provides flexibility for different research objectives .

What are the recommended applications for DDX15 antibodies?

DDX15 antibodies can be utilized across multiple experimental applications, with optimal performance parameters varying by antibody:

Researchers should conduct preliminary titration experiments to determine optimal conditions for their specific experimental systems, as performance is sample-dependent .

How can I optimize western blot protocols for DDX15 detection?

Optimizing western blot protocols for DDX15 detection requires attention to several critical parameters:

• Sample Preparation:

  • DDX15 is primarily nuclear, so nuclear extraction protocols may enhance detection

  • Validated cell lines for DDX15 detection include U-251, HeLa, U2OS, A431, NIH/3T3, and C6 cells

• Antibody Selection and Dilution:

  • For monoclonal antibodies (e.g., sc-271686): Start with 1:100-1:1000 dilution

  • For rabbit recombinant antibodies (e.g., 82137-1-RR): Use 1:5000-1:50000 dilution range

  • Consider conjugated options (HRP, fluorescent conjugates) for specialized detection methods

• Detection Optimization:

  • Expected molecular weight: 90-95 kDa

  • For infrared detection: DHX15 can be detected using IRDye 680RD secondary antibodies

  • For enhanced sensitivity: Consider antibodies conjugated to Alexa Fluor® 488, 546, 594, 647, 680, or 790

• Controls:

  • Include positive controls using validated cell lines

  • For specificity verification, consider siRNA knockdown of DHX15 in parallel samples

When troubleshooting, adjust protein loading (20-60 μg recommended), primary antibody incubation time (overnight at 4°C typically optimal), and blocking conditions to minimize background signal .

What considerations are important when using DDX15 antibodies for immunoprecipitation experiments?

Immunoprecipitation (IP) with DDX15 antibodies requires careful experimental design:

• Antibody Selection:

  • Multiple antibodies have been validated for IP, including sc-271686, ab70454, and 82137-1-RR

  • NIH/3T3 cells have been specifically validated for IP with 82137-1-RR

• Protocol Optimization:

  • Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

  • Consider crosslinking antibodies to beads to prevent antibody contamination in eluates

  • For co-immunoprecipitation of RNA-protein complexes, RNase inhibitors should be included in buffers

• Applications in RNA-Protein Interaction Studies:

  • DHX15 binds viral dsRNA and may interact with other cellular RNAs

  • RNA-immunoprecipitation can be performed with DHX15 antibodies to identify bound RNA species

  • Protein-bound RNA can be detected using infrared secondary detection (e.g., IRDye 800CW)

• Co-Immunoprecipitation Partners:

  • Known interaction partners include:

    • RIG-I (through CARDs domains)

    • MAVS (during viral infection)

    • MDA5

    • NLRP6 (in intestinal epithelial cells)

    • La/SSB autoantigen

When studying protein-protein interactions, validate antibody specificity using western blot on IP samples and consider stringency of wash conditions to preserve physiological interactions .

How can DDX15 antibodies be utilized to investigate antiviral innate immunity pathways?

DDX15 antibodies are powerful tools for exploring antiviral immunity mechanisms through several experimental approaches:

• Viral RNA Sensing and Signaling Complex Formation:

  • Use co-immunoprecipitation with DDX15 antibodies to detect interactions with:

    • RIG-I (particularly CARDs domains)

    • MAVS

    • MDA5

  • Compare complex formation under basal conditions versus viral infection or poly(I:C) stimulation

• Subcellular Localization Studies:

  • Employ immunofluorescence with DHX15 antibodies to track relocalization upon viral infection

  • Combine with markers for mitochondria (MAVS location) to verify recruitment to signaling platforms

  • Recommended antibody dilutions for IF: 1:1000-1:4000

• Functional Analysis in Knockout/Knockdown Models:

  • Use DHX15 antibodies to verify knockout/knockdown efficiency

  • Assess impact on:

    • Interferon-beta and interferon lambda-3 production

    • NLRP6 inflammasome activation

    • IL-18 secretion in intestinal epithelial cells

    • MAPK signaling activation

• RNA-Binding Assays:

  • Utilize DHX15 antibodies in RNA-immunoprecipitation experiments to identify bound viral RNAs

  • Assess DHX15's role in promoting RIG-I ATP hydrolysis in response to pathogen-associated molecular patterns (PAMPs)

This multi-faceted approach allows researchers to dissect DHX15's dual functions as both a direct viral RNA sensor and as an enhancer of RIG-I-mediated responses .

What controls should be included when studying DHX15 using antibody-based methods?

Robust controls are essential for generating reliable data with DHX15 antibodies:

• Positive Controls:

  • Cell/Tissue Selection:

    • Validated cell lines: U-251, HeLa, U2OS, A431, NIH/3T3, C6 cells

    • Tissue samples: Mouse testis tissue has been validated for IHC

  • Expression verification: DDX15 is widely expressed, but levels may vary by cell type

• Negative Controls:

  • Isotype controls: Match to antibody host species (e.g., IgG2a kappa for sc-271686)

  • Blocking peptide competition: If available, pre-incubate antibody with immunizing peptide

  • siRNA/shRNA knockdown: Create DHX15-depleted samples as specificity controls

• Technique-Specific Controls:

  • For Western blot:

    • Molecular weight markers (expected MW: 90-95 kDa)

    • Loading controls (e.g., β-actin, GAPDH)

  • For IHC/IF:

    • Secondary-only controls to assess background

    • Antigen retrieval optimization (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • For functional studies:

    • Positive controls for pathway activation (e.g., poly(I:C) for RIG-I pathway)

• Experimental Design Controls:

  • When studying viral infections, include mock-infected samples

  • For time-course experiments, include multiple timepoints to capture dynamic responses

  • For co-localization studies, include single-stained samples for compensation/bleed-through assessment

Implementing these controls ensures antibody specificity and provides context for interpreting experimental outcomes .

How do I select the appropriate DHX15 antibody for my specific research question?

Selecting the optimal DHX15 antibody requires careful consideration of several factors:

• Experimental Application Requirements:

  • For protein-protein interaction studies: Choose antibodies validated for IP (ab70454, 82137-1-RR)

  • For localization studies: Select antibodies validated for IF/ICC (sc-271686, ab254591, 82137-1-RR)

  • For quantitative analysis: Consider antibodies with wide dilution ranges for western blot (82137-1-RR, 1:5000-1:50000)

• Target Species Considerations:

  • All major antibodies react with human DHX15

  • For mouse models: sc-271686, 82137-1-RR, ab254591 are validated

  • For rat systems: sc-271686, 82137-1-RR, ab254591 are appropriate

• Epitope Location Analysis:

  • For C-terminal detection: sc-271686 targets aa 626-741

  • For N-terminal detection: ab254591 targets aa 50-150

  • Consider whether your research question involves protein domains or truncations

• Antibody Format Requirements:

  • For multiplex imaging: Consider conjugated antibodies (sc-271686 is available with various fluorescent conjugates)

  • For sensitive detection: HRP-conjugated options may provide enhanced signal

  • For specialized applications: Agarose-conjugated antibodies for direct IP

• Validation Evidence:

  • Review citations for each antibody in your application area

  • Consider antibodies with validation across multiple techniques

  • Examine published literature specific to your research focus

This strategic selection process ensures compatibility with your experimental system and research objectives .

What are methodological approaches for studying DHX15's role in RNA virus detection?

Investigating DHX15's role in viral RNA sensing requires specialized experimental approaches:

• RNA-Binding Assays:

  • RNA-immunoprecipitation with DHX15 antibodies can capture:

    • Viral dsRNA

    • Target cellular RNAs

  • Detection methods:

    • RT-PCR of specific viral RNA sequences

    • Northern blotting

    • IRDye 800CW-labeled secondary antibodies for protein-bound RNA detection

• Functional Response Assessment:

  • Measure DHX15-dependent responses following viral challenge:

    • Interferon-beta and interferon lambda-3 (IFNL3) production

    • NLRP6 inflammasome activation

    • IL-18 secretion

    • MAPK pathway activation

  • Compare wildtype vs. DHX15-depleted systems

• Mechanistic Analysis:

  • Assess DHX15's impact on RIG-I function:

    • RIG-I ATP hydrolysis assays in presence/absence of DHX15

    • Analysis of PAMP RNA recognition threshold

    • Complex formation between DHX15, RIG-I CARDs, and MAVS

• In Vivo Relevance:

  • Tissue-specific analyses:

    • Focus on intestinal epithelial cells for NLRP6 inflammasome studies

    • Examine dendritic cells for cytokine production (IFN-β, IL-6, TNFα)

  • Viral challenge models:

    • Compare susceptibility to RNA virus infection in DHX15-sufficient vs. deficient models

    • Measure viral replication kinetics

These methodological approaches allow researchers to dissect DHX15's dual function as both a direct viral RNA sensor and a coreceptor that enhances RLR signaling responses to control RNA virus infection .

How does DHX15 interact with other innate immune components in complex experimental systems?

DHX15 functions within a complex network of innate immune components, which can be studied through integrated experimental approaches:

• Interaction Network Mapping:

  • DHX15 interacts with:

    • RIG-I (through CARDs domains)

    • MAVS (following viral infection)

    • MDA5 (complementary to RIG-I interactions)

    • NLRP6 (in intestinal epithelial cells)

  • Construct interaction networks using sequential co-immunoprecipitation experiments with DHX15 antibodies

• Pathway Integration Analysis:

  • DHX15 participates in multiple pathways:

    • RLR signaling (enhancing RIG-I responses)

    • NLRP6 inflammasome activation (promoting IL-18 secretion)

    • MAPK signaling (required for cytokine production)

  • Assess pathway crosstalk through inhibitor studies and genetic approaches

• Cell-Type Specific Functions:

  • In intestinal epithelial cells: DHX15 functions with NLRP6 to restrict enteric viruses

  • In dendritic cells: DHX15 is required for IFN-β, IL-6, and TNFα production

  • Compare tissue-specific roles using cell type-specific DHX15 knockouts

• Temporal Dynamics:

  • DHX15's role may evolve during infection progression

  • Design time-course experiments to capture dynamic interactions

  • Use live-cell imaging with fluorescently-tagged DHX15 to track recruitment to signaling platforms

These approaches reveal DHX15's multifaceted role as a coreceptor that enhances innate immune signaling by lowering the threshold for RIG-I activation while simultaneously participating in parallel antiviral pathways .

What are the considerations when using DHX15 antibodies in disease models beyond viral infection?

While DHX15 is well-studied in viral immunity, its antibodies have applications in other disease contexts:

• Autoimmune Disease Research:

  • DHX15 interacts with La/SSB autoantigen, which is relevant to:

    • Systemic lupus erythematosus (SLE)

    • Sjögren's syndrome

  • Consider ANA testing context when studying DHX15 in autoimmune models

• Cancer Biology Applications:

  • DHX15's role in RNA processing may have implications in cancer

  • Validated cell lines for DHX15 detection include several cancer lines:

    • U-251 (glioblastoma)

    • HeLa (cervical cancer)

    • U2OS (osteosarcoma)

    • A431 (epidermoid carcinoma)

• Developmental Biology Research:

  • DEAD-box proteins are implicated in embryogenesis and spermatogenesis

  • DHX15 is expressed in mouse testis tissue, suitable for IHC studies

  • Consider developmental timepoints when designing experiments

• Methodological Considerations:

  • For disease models, include appropriate controls:

    • Age-matched controls (ANA positivity increases with age)

    • Treatment controls (medications can influence ANA results)

    • Cell-type specific markers to confirm DHX15 expression patterns

When applying DHX15 antibodies to novel disease models, researchers should first validate antibody performance in their specific experimental system and carefully consider disease-relevant controls .

What emerging techniques might enhance DHX15 research beyond traditional antibody applications?

Several cutting-edge approaches can complement traditional antibody-based methods for DHX15 research:

• Advanced Imaging Techniques:

  • Super-resolution microscopy can reveal DHX15's precise subcellular localization

  • Live-cell imaging with fluorescently-tagged DHX15 can track dynamic recruitment

  • Proximity ligation assays can visualize protein-protein interactions in situ

  • Consider fluorescent conjugates (Alexa Fluor® 488, 546, 594, 647, 680, 790) for multiplexed imaging

• Proteomics Approaches:

  • IP-mass spectrometry using DHX15 antibodies can identify novel interaction partners

  • Crosslinking mass spectrometry can map DHX15 binding interfaces

  • Phosphoproteomics can reveal regulation of DHX15 function by post-translational modifications

• Genomic Technologies:

  • CRISPR-Cas9 gene editing to create DHX15 mutants for structure-function studies

  • RNA-seq of DHX15-depleted cells to identify regulated transcripts

  • ChIP-seq could explore potential roles in transcriptional regulation

• Structural Biology Integration:

  • Antibody epitope mapping to relate functional outcomes to structural domains

  • Cryo-EM studies of DHX15-containing complexes (spliceosome, RIG-I complexes)

  • Single-molecule studies of DHX15's RNA helicase activity

These emerging techniques can provide deeper insights into DHX15's dual functions in RNA processing and innate immunity, potentially revealing new therapeutic targets for viral infections and related disorders .