DDX39A Antibody

What is DDX39A and what cellular functions does it perform?

DDX39A is a member of the DEAD box protein family characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD) and functions as an RNA helicase. These proteins are involved in numerous cellular processes requiring alteration of RNA secondary structure, including translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly . DDX39A has both cytoplasmic and nuclear localization , with recent studies demonstrating its role in mRNA export, innate immunity, and potentially cancer progression . Developmentally, DEAD box proteins like DDX39A may be involved in embryogenesis, spermatogenesis, and cellular growth and division .

What applications are DDX39A antibodies validated for?

The DDX39A Polyclonal Antibody (E-AB-52576) is primarily validated for immunohistochemistry (IHC) applications with a recommended dilution range of 1:50-1:200 . This rabbit-derived polyclonal antibody has been specifically verified with human breast cancer and thyroid cancer samples . The antibody demonstrates reactivity with human, mouse, and rat samples, making it versatile for comparative studies across these species .

How should DDX39A antibodies be stored and handled?

For optimal antibody performance, DDX39A antibodies should be stored at -20°C where they remain valid for approximately 12 months . It's critical to avoid freeze/thaw cycles which can degrade antibody quality . The antibody is typically shipped with ice packs and should be stored immediately at the recommended temperature upon receipt . The DDX39A Polyclonal Antibody is provided at a concentration of 1.02 mg/mL in a phosphate buffered solution (pH 7.4) containing 0.05% stabilizer and 50% glycerol .

How does DDX39A contribute to antiviral immunity?

Recent genetic screening has identified DDX39A as an antiviral factor against multiple alphaviruses including chikungunya virus (CHIKV), Sindbis virus (SINV), Venezuelan equine encephalitis virus (VEEV), and O'nyong-nyong virus (ONNV) . Upon infection, the predominantly nuclear DDX39A accumulates in the cytoplasm where it inhibits alphavirus replication independent of the canonical interferon pathway . Mechanistically, DDX39A binds to the 5' conserved sequence element (5'CSE) in alphavirus genomic RNA . This element is essential for the antiviral activity of DDX39A, as loss of this structure renders CHIKV insensitive to DDX39A's inhibitory effects .

What is the specificity of DDX39A's antiviral activity?

DDX39A demonstrates remarkable specificity in its antiviral activity. Research has shown that while DDX39A effectively controls diverse alphaviruses, it does not impact infection with other positive-sense RNA viruses such as coronaviruses (229E, OC43) and picornaviruses (coxsackie B virus) . Similarly, DDX39A does not affect infection with negative-sense RNA viruses including rhabdoviruses (vesicular stomatitis virus) and bunyaviruses (Rift Valley Fever virus) . This specificity suggests DDX39A recognizes structural elements unique to alphavirus genomes.

How does DDX39A differ from its paralog DDX39B in viral infections?

Despite sharing approximately 90% homology with its paralog DDX39B, DDX39A exhibits distinct functional properties during viral infection . Experimental evidence demonstrates that knockdown of DDX39A, but not DDX39B, results in increased alphavirus infection . This functional divergence despite high sequence similarity makes DDX39A particularly interesting for studying protein specialization in antiviral immunity.

How does SUMOylation affect DDX39A function?

DDX39A is subject to post-translational modification by Small Ubiquitin-like Modifier (SUMO) proteins. Research has identified SUMO1, SUMO2, and SUMO3 modifications on immunoprecipitated DDX39A . Notably, during vesicular stomatitis virus infection, specifically the SUMO1 modification of DDX39A decreases . RanBP2 acts as an E3 SUMO ligase for DDX39A, enhancing SUMO1 modification which attenuates DDX39A's RNA binding ability . This regulation presents a sophisticated mechanism where viral infection reduces SUMO1 modification of DDX39A, enhancing its binding to specific innate immunity-associated mRNAs .

How does DDX39A regulate host mRNA export during viral infection?

Studies have revealed that human DDX39A can facilitate RNA virus escape from innate immunity by trapping specific mRNAs encoding antiviral signaling components (TRAF3, TRAF6, and MAVS) in the nucleus . During viral infection, reduced SUMOylation of DDX39A enhances its ability to bind these innate immunity-associated mRNAs, effectively sequestering them and limiting their expression . This mechanism represents a way that viruses might exploit DDX39A to evade host immune responses, presenting a contrast to DDX39A's direct antiviral activity against alphaviruses .

What experimental approaches can be used to study DDX39A's interaction with viral RNA?

Researchers have successfully employed cross-linking immunoprecipitation followed by sequencing (CLIP-Seq) to map DDX39A's interaction with viral RNA, revealing preferential binding to the 5'CSE element of CHIKV RNA . Other effective experimental approaches include:

• RNAi screening to identify DDX39A's role in viral infection

• CRISPR-Cas9 knockout for validation studies

• RT-qPCR to quantify viral RNA levels

• Immunoblotting to detect protein expression

• Automated microscopy to assess infection levels

• Viral titration assays to measure infectious particle production

These methods collectively provide a comprehensive toolkit for investigating DDX39A's interactions with viral components and its functional consequences.

What is the role of DDX39A in cancer research?

Beyond its antiviral functions, DDX39A is thought to play a role in cancer progression and patient prognosis, particularly in gastrointestinal stromal tumors . The availability of DDX39A antibodies verified for use with human breast cancer and thyroid cancer samples supports investigation into its roles across different cancer types . The involvement of DDX39A in fundamental RNA processing mechanisms suggests it could influence expression of genes relevant to oncogenesis and tumor progression, positioning it as a potential biomarker or therapeutic target.

How can researchers validate DDX39A antibody specificity?

For rigorous DDX39A research, antibody validation is crucial. Researchers should:

• Perform siRNA or CRISPR knockdown/knockout experiments to confirm antibody specificity by demonstrating reduced or absent signal in depleted samples

• Include positive controls from tissues known to express DDX39A (such as breast or thyroid cancer samples)

• Validate across multiple techniques (IHC, Western blot, immunofluorescence) when possible

• Test reactivity in multiple species if conducting comparative studies

The research cited in the search results demonstrates validation approaches including siRNA depletion with immunoblot confirmation and CRISPR-Cas9 knockout verification , providing methodological templates for thorough validation.

What are the best practices for optimizing IHC protocols with DDX39A antibodies?

For optimal immunohistochemistry results with DDX39A antibodies:

• Begin with the recommended dilution range (1:50-1:200) and perform titration experiments to determine optimal concentration for specific tissues

• Include positive control tissues (breast cancer or thyroid cancer samples have been verified)

• Consider antigen retrieval methods appropriate for nuclear proteins

• Implement appropriate blocking steps to minimize background staining

• When studying DDX39A localization during viral infection, compare infected versus uninfected samples to observe cytoplasmic relocalization

• Document both nuclear and cytoplasmic staining patterns, as DDX39A exhibits dual localization

How can researchers distinguish between DDX39A and its homologous proteins?

Given the high homology (~90%) between DDX39A and DDX39B , researchers must employ strategies to ensure specificity:

• Select antibodies raised against unique epitopes not shared with DDX39B or other DEAD-box helicases

• Perform parallel experiments with specific siRNAs targeting DDX39A versus DDX39B to confirm differential effects

• Consider using tagged constructs for overexpression studies to distinguish the proteins

• When possible, employ mass spectrometry-based approaches for definitive protein identification

• Include appropriate controls in functional assays to differentiate DDX39A-specific effects from those of related proteins