DYRK4 Antibody

What is DYRK4 and what applications are DYRK4 antibodies validated for?

DYRK4 belongs to a conserved family of serine/threonine protein kinases that function in the regulation of cell differentiation, proliferation, survival, and development . It is thought to be a testis-specific kinase predominantly expressed in the testis, though Dyrk4-deficient mice are fertile, suggesting functional redundancy with other DYRKs . Recent research has revealed DYRK4's critical role in antiviral immunity .

DYRK4 antibodies have been validated for several applications:

The choice of application should be guided by your specific research question and experimental design .

What is the species reactivity of available DYRK4 antibodies?

Different commercial DYRK4 antibodies show varying species reactivity profiles:

When selecting an antibody, it's crucial to verify reactivity with your species of interest. For cross-species studies, Proteintech's antibody offers broader reactivity . Always validate the antibody in your specific experimental system before proceeding with full-scale experiments.

How should DYRK4 antibodies be stored and handled?

Proper storage and handling are critical for maintaining antibody performance:

• Storage temperature: Most DYRK4 antibodies should be stored at -20°C

• Buffer conditions: Most are supplied in PBS with glycerol and sodium azide

• Aliquoting: For Abbexa antibodies, aliquoting is recommended to avoid repeated freeze/thaw cycles

• Stability: When stored properly, antibodies are typically stable for at least one year after shipment

Proteintech specifically notes that "Aliquoting is unnecessary for -20°C storage" for their product, which differs from some other manufacturers' recommendations .

How does DYRK4 regulate antiviral immune responses?

Recent research has revealed DYRK4 as an essential regulator of virus-triggered activation of IRF3 and NF-κB, type I interferon induction, and cellular antiviral responses . The mechanism involves:

• Upregulation of DYRK4 mRNA and protein levels during viral infection (SeV, VSV, or HSV-1)

• Function as a scaffold protein, recruiting TRIM71 and LUBAC to IRF3

• Promotion of IRF3 linear ubiquitination, maintaining IRF3 stability and activation during viral infection

• Enhancement of IRF3-mediated antiviral response

Notably, Dyrk4-knockout mice show increased susceptibility to viral infection, with:

• Higher mortality rates when infected with VSV

• Reduced mRNA expression of Ifnb1, Cxcl10, Isg15, and Il6 in spleen, lung, and liver tissues

• Lower levels of IFN-β and CXCL10 in sera

• Increased viral replication in liver and lung

• Greater lung tissue damage following VSV infection

These findings establish DYRK4 as a key positive regulator of antiviral innate immunity.

What is known about DYRK4 splice variants and their functional differences?

DYRK4 undergoes complex alternative splicing that affects both function and localization:

• N-terminal variants: Human DYRK4 exists in multiple isoforms with different N-terminal regions:

  • A 520-amino acid isoform (NM_003845)

  • A 635-amino acid isoform (AK308260)

  • A 644-amino acid isoform (includes an extra exon encoding 9-10 additional amino acids)

• Variants affecting subcellular localization: The longer isoform contains a nuclear localization signal (NLS) in its extended N-terminus that:

  • Mediates interaction with importin α3 and α5

  • Can target heterologous proteins to the nucleus

  • Results in different nucleocytoplasmic mobility compared to shorter isoforms

• Variants affecting catalytic activity:

  • Exclusion of exon 18 leads to a frameshift and truncated kinase domain, predicted to lack activity

  • Alternative splicing of exon 19 results in inclusion/exclusion of three amino acids (CLV) in kinase subdomain XI, significantly affecting enzymatic activity

• Tissue-specific expression patterns: The splice variants show distinct tissue-specific expression patterns, suggesting specialized functions in different cellular contexts .

Understanding which splice variant you are detecting is crucial when interpreting experimental results with DYRK4 antibodies.

Is DYRK4's role in antiviral signaling dependent on its kinase activity?

Interestingly, research indicates that DYRK4's role in virus-triggered signaling is independent of its kinase activity . Evidence supporting this includes:

• Kinase-inactive mutants of DYRK4 (K133R, Y264F, and K133R/Y264F double mutants) still enhanced SeV-induced activation of the IFNβ promoter

• These kinase-inactive mutants promoted SeV-induced phosphorylation of IRF3 and IκBα

• DYRK4 did not promote IFN-γ-induced IRF1 transcription, suggesting pathway specificity

This contrasts with other DYRK family members like DYRK2, whose functions often depend on kinase activity . Instead, DYRK4 appears to function primarily as a scaffold protein in antiviral signaling, recruiting proteins like TRIM71 and LUBAC to IRF3 .

What controls should be included when using DYRK4 antibodies in experimental procedures?

For rigorous experimental design with DYRK4 antibodies, include these controls:

• Positive controls:

  • For Western blot: Use validated lysates from tissues/cells known to express DYRK4, such as:

    • Mouse lung tissue

    • Human brain tissue

    • Jurkat cells

    • Mouse testis tissue

    • U-937 cells

  • For IHC: Human lymphoma tissue has been validated for some antibodies

• Negative controls:

  • Samples from DYRK4 knockout models (cells or tissues)

  • Primary antibody omission control

  • Isotype control (rabbit IgG)

• Validation controls:

  • Peptide competition assay using the immunizing peptide

  • siRNA/shRNA knockdown samples with validated reduction in DYRK4 expression

  • Different antibodies targeting distinct epitopes of DYRK4

• Loading/processing controls:

  • Housekeeping protein detection (e.g., GAPDH, β-actin)

  • For phosphorylation studies, total DYRK4 levels should be measured alongside phosphorylated forms

When using shRNA targeting DYRK4, researchers have successfully employed the pLKO.1-TCR cloning vector transfected with PEI, followed by confirmation via immunoblotting or qPCR analysis .

How can I verify the specificity of a DYRK4 antibody?

Verification of antibody specificity is critical for reliable results. Consider these approaches:

• Genetic models:

  • Test antibody using DYRK4 knockout cell lines or tissues

  • HEK293T cells with DYRK4 knockout and A549 cells with DYRK4 knockdown have been successfully created using CRISPR/Cas9 technology

  • Use validated sgRNA sequences available in published literature

• Multiple detection methods:

  • Compare results across multiple applications (WB, IHC, IF)

  • Use multiple antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

• Molecular validation:

  • Verify the correct molecular weight (~60 kDa for the main isoform)

  • Be aware that DYRK4 has multiple isoforms with different molecular weights (59, 72, and 73 kDa)

  • Consider the impact of post-translational modifications

• Correlation with functional data:

  • Correlate antibody detection with expected biological responses (e.g., changes during viral infection)

  • Validate using overexpression systems with tagged DYRK4 constructs

For CRISPR/Cas9-mediated knockout validation, researchers have utilized the lenti-CRISPR/Cas9-v2 system with packaging plasmids pMD2.G and psPAX2, followed by puromycin selection for 5-7 days .

What techniques are recommended for studying DYRK4 interactions with other proteins?

Based on successful approaches in the literature, several techniques are effective for studying DYRK4 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; 10 mM NaF; 1 mM Na3VO4; and 2 mM DTT) containing protease inhibitors

  • Centrifuge lysates at 12,000 rpm for 10 min at 4°C

  • Incubate with anti-Flag beads or specific antibodies plus protein A/G beads overnight at 4°C

  • Wash three times with buffer before elution and immunoblot analysis

• Mass spectrometry for interaction partner identification:

  • Transfect cells with tagged DYRK4

  • Purify using appropriate beads

  • Separate by SDS-PAGE and stain with Coomassie blue

  • Cut deeper color protein bands for MS analysis

• Confocal microscopy for colocalization studies:

  • This approach has successfully demonstrated DYRK4 colocalization with IRF3

  • Consider that DYRK4 is predominantly distributed in the cytoplasm and to a lesser extent in the nucleus

• In vitro pull-down assays:

  • Use recombinant DYRK4 proteins

  • Bacterially expressed GST-DYRK4 can be obtained by inducing transformed E. coli BL21(DE3)pLysS with 0.1 mM IPTG for 4–6 h at 20°C

  • Alternatively, recombinant human GST-DYRK4 expressed in insect cells has been used successfully

• Functional validation methods:

  • Reporter assays with IFNβ, ISRE, and NF-κB promoters have demonstrated DYRK4's interactions within signaling pathways

  • Overexpression or knockdown approaches followed by pathway activation assessment

Why might I observe multiple bands in a Western blot using DYRK4 antibodies?

Multiple bands in DYRK4 Western blots can result from several biological and technical factors:

• Multiple isoforms: DYRK4 has at least 5 documented isoforms with molecular weights ranging from 59-73 kDa :

  • 520 amino acid isoform (~60 kDa)

  • 635 amino acid isoform (~72 kDa)

  • 644 amino acid isoform (~73 kDa)

  • Additional splice variants affecting exon 18 and exon 19

• Tissue-specific expression patterns: Different tissues express different isoforms, which may explain variation in banding patterns across sample types .

• Post-translational modifications:

  • Phosphorylation (DYRK4 undergoes autophosphorylation)

  • Ubiquitination (DYRK4 interacts with ubiquitin machinery)

  • Other modifications that alter apparent molecular weight

• Degradation products: Particularly in samples with suboptimal storage or preparation.

To distinguish between these possibilities:

• Compare with known positive controls

• Use tissue or cell types with documented DYRK4 expression patterns

• Consider using isoform-specific antibodies if available

• Include phosphatase treatment to identify bands resulting from phosphorylation

How do splice variants of DYRK4 affect experimental design and data interpretation?

The presence of multiple DYRK4 splice variants has significant implications for experimental design:

• Antibody epitope location: Consider whether your antibody recognizes:

  • A specific N-terminal variant (if the epitope is in this region)

  • All isoforms (if the epitope is in a conserved region)

  • For example, the Abbexa antibody targets a synthetic peptide between 455-485 amino acids from the C-terminal region

• Functional differences between variants:

  • Nuclear vs. cytoplasmic localization (longer variants contain NLS)

  • Differences in kinase activity (variants lacking the CLV motif in subdomain XI show reduced activity)

  • Tissue-specific expression patterns

• Detection methods optimization:

  • For RT-PCR analysis, use primers that can distinguish between splice variants

  • For protein analysis, consider gradient gels to better separate closely migrating isoforms

• Experimental interpretation:

  • Be precise about which isoform(s) you are studying

  • Consider isoform-specific functions when interpreting results

  • Be aware that knockout/knockdown approaches may affect multiple isoforms differently

Researchers studying specific splice variants have successfully used isoform-specific primer sets for RT-PCR analysis (sequences available in supplemental tables of relevant publications) .

How should I interpret DYRK4 expression changes during viral infection?

When analyzing DYRK4 expression changes during viral infection:

• Normal expression dynamics:

  • DYRK4 mRNA and protein levels typically increase during infection with RNA viruses (SeV, VSV) and DNA viruses (HSV-1)

  • This upregulation begins within hours of infection

• Functional correlation:

  • Increased DYRK4 expression correlates with enhanced IRF3 and NF-κB activation

  • It promotes IFNβ induction and antiviral responses

  • The recruitment of TRIM71 and LUBAC to IRF3 increases with viral infection

• Interpretation framework:

  • Assess both mRNA and protein levels

  • Correlate with activation of downstream pathways (IRF3 phosphorylation, IFNβ induction)

  • Compare across multiple timepoints to capture the dynamic response

  • Consider cell type-specific responses (BMDMs, BMDCs, MLFs show different patterns)

• Control considerations:

  • Include uninfected controls

  • Use multiple virus types to distinguish virus-specific vs. general antiviral responses

  • Consider kinetics of expression changes (early vs. late response)

When studying viral infection models, researchers have successfully employed various experimental systems:

• In vitro: THP-1 cells, HEK293T cells, BMDMs, BMDCs, MLFs

• In vivo: VSV infection of Dyrk4+/+ and Dyrk4-/- mice via tail vein injections

What are emerging areas of DYRK4 research beyond antiviral immunity?

While recent research has focused on DYRK4's role in antiviral immunity, several emerging areas warrant further investigation:

• Cancer biology: Preliminary evidence suggests potential oncogenic roles:

  • A tumorigenic chimeric transcript, RAD51AP1-DYRK4, enhances MEK/ERK signaling activation

  • This chimeric protein increases invasiveness of ductal mammary carcinomas

  • Hypomethylation of DYRK4 in peripheral blood is associated with increased lung cancer risk

• Substrate specificity: DYRK4 shows distinct substrate preferences compared to other DYRK family members:

  • Unlike DYRK1A, DYRK4 does not show strong preference for substrates with arginine at position P-3

  • This suggests unique targets and functions compared to other family members

• Testis function: Originally identified as a testis-specific kinase:

  • Despite predominant testis expression, Dyrk4-deficient mice are fertile

  • This suggests functional redundancy with other DYRKs in testicular development

  • The specific role in testicular function remains to be fully characterized

• Neural functions: With expression detected in brain tissue, potential neurological roles remain unexplored

• Therapeutic targeting: Given its role in antiviral immunity, DYRK4 represents a potential therapeutic target:

  • Enhancing DYRK4 function might boost antiviral responses

  • Targeting specific isoforms could provide selective intervention strategies

Future research should employ isoform-specific approaches and consider the diverse functional capacities of different DYRK4 variants across biological contexts.