Phospho-DDX3X (T322) Antibody

What is DDX3X and why is phosphorylation at T322 significant?

DDX3X is a multifunctional ATP-dependent RNA helicase belonging to the DEAD-box protein family, defined by the presence of the conserved Asp-Glu-Ala-Asp (DEAD) motif. It plays crucial roles in multiple cellular processes including transcription regulation, translation initiation, and RNA metabolism .

Phosphorylation at threonine 322 (T322) represents a specific post-translational modification that may regulate DDX3X function. This phosphorylation site is of particular interest because:

• It may modulate DDX3X's helicase or ATPase activity

• It potentially affects DDX3X's interaction with other cellular proteins

• It might be involved in cellular stress responses and stress granule formation

• It could be dysregulated in pathological conditions including neurodevelopmental disorders and cancer

Understanding this specific phosphorylation provides insights into regulatory mechanisms controlling DDX3X activity in both normal cellular processes and disease states .

What are the key characteristics of Phospho-DDX3X (T322) antibodies?

Phospho-DDX3X (T322) antibodies are specifically designed to detect DDX3X protein only when phosphorylated at threonine 322. Key characteristics include:

These antibodies are purified through affinity chromatography using epitope-specific immunogen to ensure high specificity for the phosphorylated form .

How should Phospho-DDX3X (T322) antibody be optimized for immunohistochemistry?

For optimal immunohistochemistry (IHC) results with Phospho-DDX3X (T322) antibody, follow these methodological recommendations:

• Dilution Range: Use at 1:100-1:300 dilution as a starting point for optimization

• Antigen Retrieval:

  • High-pressure and high-temperature retrieval with Tris-EDTA buffer (pH 8.0) has shown effectiveness

  • Alternative: Citrate buffer (pH 6.0) may work for some tissue types

• Incubation Parameters:

  • Temperature: 4°C

  • Duration: Overnight incubation recommended for optimal signal-to-noise ratio

• Controls:

  • Positive Control: Human brain tissue has been validated

  • Negative Control: Pre-absorption with immunogen peptide to confirm specificity

• Detection System:

  • Use a detection system appropriate for rabbit or mouse IgG depending on the host species of your antibody

  • Minimize background by adjusting blocking conditions (5% normal serum from secondary antibody host species)

• Signal Validation: Consider phosphatase treatment of parallel sections to confirm phospho-specificity

Experimental evidence shows successful IHC application in human brain tissue, with clear nuclear and cytoplasmic staining patterns that are abolished by pre-absorption with the phosphopeptide .

What are the optimal conditions for immunofluorescence experiments using this antibody?

For optimal immunofluorescence (IF) results with Phospho-DDX3X (T322) antibody:

• Dilution Optimization:

  • Recommended range: 1:200-1:1000

  • Begin at 1:500 and adjust based on signal intensity

• Cell Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde (15 minutes at room temperature)

  • Permeabilize with 0.2% Triton X-100 in PBS (10 minutes)

  • Blocking: 5% normal serum in PBS (1 hour at room temperature)

• Antibody Incubation:

  • Primary antibody: Incubate overnight at 4°C

  • Wash 3× with PBS + 0.1% Tween-20

  • Secondary antibody: 1-2 hours at room temperature (fluorophore-conjugated anti-rabbit or anti-mouse IgG depending on primary antibody host)

• Signal Verification:

  • Perform phosphopeptide blocking control by pre-incubating antibody with immunizing phosphopeptide

  • Include known stimulation control (e.g., serum treatment at 20% for 30 minutes has been shown to increase phosphorylation)

  • Consider co-staining with total DDX3X antibody to assess phosphorylation ratio

• Subcellular Localization Analysis:

  • Counterstain nuclei with DAPI

  • Consider co-staining with markers of stress granules (e.g., TIA1) if studying stress responses

Published research has successfully applied this antibody in HUVEC cells following serum treatment, with specific staining abolished by phosphopeptide pre-absorption .

How can specificity for phosphorylated T322 versus unphosphorylated DDX3X be verified?

Verifying the specificity of Phospho-DDX3X (T322) antibody is crucial for experimental rigor. Implement these methodological approaches:

• Phosphopeptide Competition Assay:

  • Pre-incubate antibody with the phosphorylated peptide used as immunogen

  • In parallel, pre-incubate with the corresponding non-phosphorylated peptide

  • The phosphopeptide should completely abolish signal, while non-phospho peptide should have minimal effect

• Phosphatase Treatment Control:

  • Treat half of your sample with lambda phosphatase before antibody incubation

  • Signal should be significantly reduced in phosphatase-treated samples

• Phospho-ELISA Validation:

  • Enzyme-Linked Immunosorbent Assay using both phosphopeptide and non-phosphopeptide

  • Results should show strong reactivity with phosphopeptide and minimal reactivity with non-phosphopeptide

• Stimulation-Dependent Phosphorylation:

  • Compare cells under basal conditions versus stimulated conditions known to induce T322 phosphorylation

  • Serum treatment (20% for 30 minutes) has been demonstrated to increase T322 phosphorylation in HUVEC cells

• Genetic Controls:

  • Use cells with DDX3X knockdown or knockout as negative controls

  • Consider T322A mutant expression to create a non-phosphorylatable control

Experimental data demonstrates successful application of phospho-ELISA showing strong differential detection between phospho and non-phospho peptides, confirming antibody specificity .

What are the common pitfalls when working with Phospho-DDX3X (T322) antibody and how can they be avoided?

Working with phospho-specific antibodies presents unique challenges. Here are methodological solutions for common pitfalls:

• False Negative Results:

  • Cause: Rapid dephosphorylation during sample preparation

  • Solution: Add phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate) to all buffers; maintain samples at 4°C throughout processing

• Weak or Absent Signal:

  • Cause: Low phosphorylation levels under basal conditions

  • Solution: Consider treating cells with appropriate stimuli (serum, growth factors); optimize antibody concentration; increase antigen retrieval stringency; extend primary antibody incubation time

• High Background:

  • Cause: Non-specific binding or insufficient blocking

  • Solution: Increase blocking time (5% BSA or normal serum); optimize antibody dilution; ensure thorough washing steps; pre-absorb antibody with non-phospho protein to remove any antibodies recognizing unphosphorylated epitopes

• Cross-Reactivity Issues:

  • Cause: Antibody recognizing similar phosphorylated epitopes in other proteins

  • Solution: Validate with specific controls (DDX3X knockout/knockdown); always perform phosphopeptide competition controls

• Storage-Related Degradation:

  • Cause: Repeated freeze-thaw cycles

  • Solution: Aliquot antibody upon receipt; store at -20°C; for frequent use, keep a working aliquot at 4°C for up to one month

• Inconsistent Results Between Techniques:

  • Cause: Different sample preparation affecting phosphorylation preservation

  • Solution: Standardize sample preparation protocols; consider technique-specific optimizations while maintaining phosphorylation state

Rigorous experimental controls are essential for validating results with phospho-specific antibodies, as demonstrated by the successful use of peptide competition assays in published immunofluorescence and immunohistochemistry applications .

How can Phospho-DDX3X (T322) antibody be used to study stress granule dynamics and DDX3X mutant behavior?

Phospho-DDX3X (T322) antibody offers a powerful tool for investigating stress granule (SG) dynamics and the impact of DDX3X mutations, particularly in neurological disorders:

• Stress Granule Formation Analysis:

  • Methodology: Treat cells with stress inducers (sodium arsenite, heat shock, oxidative stress) and monitor phospho-DDX3X localization

  • Analysis: Quantify co-localization with known SG markers (TIA1, G3BP1) using confocal microscopy and image analysis software

  • Significance: Determines if T322 phosphorylation affects DDX3X recruitment to SGs

• Mutant DDX3X Behavior Assessment:

  • Approach: Compare phosphorylation patterns between wild-type DDX3X and disease-associated mutants (e.g., R376C, L556S)

  • Technical Setup: Create stable cell lines expressing wild-type or mutant DDX3X and assess T322 phosphorylation levels

  • Research Question: Do disease-causing mutations alter phosphorylation at T322?

• SG Dynamics Quantification:

  • Live Cell Analysis: Track SG mobility using time-lapse imaging of cells expressing fluorescently-tagged DDX3X variants

  • Measurement Parameters: Calculate displacement, coalescence rates, and dissolution kinetics

  • Correlation Analysis: Assess how T322 phosphorylation status correlates with SG mobility metrics

• Liquid-Solid Phase Transition Studies:

  • Experimental Approach: Treat SGs containing phospho-DDX3X with 1,6-hexanediol to disrupt weak hydrophobic interactions

  • Comparative Analysis: Assess differential sensitivity of wild-type versus mutant phospho-DDX3X-containing granules

  • Physiological Relevance: Determines if phosphorylation affects phase separation properties relevant to neurodegenerative processes

Research has demonstrated that DDX3X mutations associated with intellectual disability (ID) show differential SG assembly and aggregation propensity. Specifically, the L556S mutant forms dense cytoplasmic granules under normal conditions, while R376C shows diffuse localization. When stressed with sodium arsenite, both mutants form SGs with reduced mobility compared to wild-type, suggesting potential protein aggregation within SGs containing mutant DDX3X .

What is the role of T322 phosphorylation in DDX3X-mediated RNA metabolism and translation regulation?

The phosphorylation of DDX3X at T322 may significantly impact its function in RNA metabolism and translation regulation, presenting important research directions:

• Translation Initiation Studies:

  • Methodological Approach: Polysome profiling with phospho-mimetic (T322D/E) and phospho-deficient (T322A) DDX3X mutants

  • Measurement Parameter: Assess changes in translation efficiency of specific mRNA targets

  • Technical Tools: Combine with RNA immunoprecipitation (RIP) to identify differentially bound RNAs based on phosphorylation status

• RNA-Protein Interaction Analysis:

  • Experimental Design: Compare RNA binding profiles of phosphorylated versus non-phosphorylated DDX3X

  • Techniques: RNA electrophoretic mobility shift assay (EMSA) with recombinant proteins

  • Focus Area: Determine if T322 phosphorylation affects binding to RNA G-quadruplex (rG4) structures, particularly in 5'-UTRs of specific mRNAs like NRAS

• ATPase and Helicase Activity Assessment:

  • In vitro Enzymatic Assays: Compare ATPase and helicase activities of phosphorylated, non-phosphorylated, and phospho-mimetic DDX3X variants

  • Substrate Specificity: Test activity on various RNA structures (dsRNA, ssRNA with overhangs, G-quadruplexes)

  • Kinetic Analysis: Determine if phosphorylation alters enzyme kinetics (Km, Vmax, processivity)

• Post-transcriptional Regulation Pathways:

  • Research Direction: Investigate how T322 phosphorylation affects DDX3X's role in:

    • mRNA export from nucleus to cytoplasm

    • Cytoplasmic mRNP (messenger ribonucleoprotein) complex formation

    • Stress granule assembly and dynamics

    • miRNA processing and function

• Signaling Pathway Integration:

  • Kinase Identification: Determine which kinase(s) phosphorylate DDX3X at T322

  • Pathway Mapping: Establish the signaling cascades that regulate this phosphorylation event

  • Physiological Triggers: Identify cellular conditions that modulate T322 phosphorylation levels

DDX3X is known to bind RNA G-quadruplex structures, including those in the 5'-UTR of NRAS mRNA, and is involved in regulating translation initiation. Understanding how T322 phosphorylation affects these functions may provide insights into both normal cellular processes and disease mechanisms, particularly in neurodevelopmental disorders and cancer .

How can Phospho-DDX3X (T322) antibody be used to study the relationship between DDX3X mutations and intellectual disability?

Phospho-DDX3X (T322) antibody provides a valuable tool for investigating the mechanistic link between DDX3X mutations and intellectual disability (ID):

• Mutation-Phosphorylation Relationship Analysis:

  • Experimental Approach: Compare T322 phosphorylation levels in patient-derived cells or in vitro models expressing ID-associated DDX3X mutations

  • Cell Models: Patient-derived fibroblasts, iPSC-derived neurons, or transfected neural progenitor cells

  • Hypothesis Testing: Determine if ID-associated mutations alter T322 phosphorylation patterns

• Genotype-Phenotype Correlation Studies:

  • Cohort Analysis: Examine phosphorylation patterns across samples from patients with different mutation types:

    • Missense mutations (particularly recurrent mutations at R488, R376, T532M, R326, I415)

    • Nonsense/frameshift (loss-of-function) mutations

    • Splice site mutations

  • Clinical Correlation: Associate phosphorylation patterns with severity of clinical features (PMG, seizures, microcephaly)

• Neurodevelopmental Process Investigation:

  • Developmental Timing: Monitor T322 phosphorylation during neural differentiation and cortical development

  • Cellular Processes: Assess impact on neurogenesis, neuronal migration, and neurite outgrowth

  • Signaling Pathway Analysis: Examine interaction with pathways critical for brain development

• Therapeutic Target Identification:

  • Phosphorylation Modulation: Test compounds that affect T322 phosphorylation levels

  • Functional Rescue: Assess if normalizing phosphorylation patterns rescues cellular phenotypes

  • Screening Platform: Develop high-throughput assays using the antibody to identify potential therapeutic compounds

Research has identified 107 individuals with DDX3X mutations, with striking correlations between specific mutations and clinical outcomes. Notably, missense mutations are significantly more likely to be associated with polymicrogyria (PMG) and severe phenotypes, while nonsense/frameshift mutations never presented with PMG. Recurrent mutations at specific sites (R488, R376, T532M, R326, I415) resulted in similar phenotypes, suggesting site-specific functional consequences .

What is the relationship between DDX3X T322 phosphorylation and stress granule dynamics in neurological disorders?

The relationship between DDX3X T322 phosphorylation and stress granule (SG) dynamics represents an important area of investigation in neurological disorders:

• Stress Granule Formation and Clearance Assessment:

  • Methodological Approach: Compare phospho-T322 DDX3X localization in SGs between normal and disease models

  • Quantitative Parameters: Measure SG size, number, persistence time, and phospho-DDX3X content

  • Technical Setup: Use time-lapse imaging with stress induction (sodium arsenite, heat shock at 43°C)

• Mutant-Specific SG Behavior Analysis:

  • Experimental Design: Compare SG dynamics in cells expressing wild-type versus ID-associated mutant DDX3X

  • Observations from Literature:

    • L556S mutant forms dense cytoplasmic granules even under unstressed conditions (45% of cells)

    • R376C shows diffuse localization similar to wild-type under basal conditions

    • Both mutations show altered SG dynamics following stress induction

• Phase Separation Properties Investigation:

  • Approach: Assess liquid-like versus solid-like properties of SGs containing phospho-T322 DDX3X

  • Key Finding: Mutant DDX3X-containing SGs show reduced mobility following stress treatment (60 minutes after sodium arsenite exposure)

  • Mechanistic Test: 1,6-hexanediol treatment disrupts liquid-like granules but not solid aggregates, providing insight into SG material properties

• Cell Viability and Neurotoxicity Correlation:

  • Assays: MTS cell viability and TUNEL apoptosis detection

  • Research Finding: Expression of L556S mutant reduces cell viability and increases apoptosis compared to wild-type DDX3X

  • Relevance: Links aberrant SG dynamics to cellular toxicity and potential neurodegeneration

• RNA Metabolism Disruption Assessment:

  • Hypothesis: Altered phosphorylation affects DDX3X's RNA helicase function, disrupting RNA metabolism

  • Approach: RIP-seq to identify differentially bound RNAs between wild-type and mutant DDX3X

  • Correlation Analysis: Associate phosphorylation status with RNA binding patterns and processing efficiency

The experimental evidence shows that ID-linked missense mutations disrupt RNA helicase activity and induce stress granule formation in neural progenitors and neurons. Particularly, L556S and R376C mutations show differential effects on SG dynamics, with distinct mobility characteristics after stress induction, suggesting a potential mechanism for neurological dysfunction .

What kinases and phosphatases regulate DDX3X T322 phosphorylation and how might this be therapeutically targeted?

Identifying the regulatory enzymes controlling DDX3X T322 phosphorylation opens avenues for therapeutic intervention:

• Kinase Identification Strategy:

  • Bioinformatic Approach: Analyze the sequence context around T322 for kinase consensus motifs

  • Kinase Inhibitor Screening: Systematic testing of specific kinase inhibitors to identify which reduces T322 phosphorylation

  • In vitro Kinase Assays: Recombinant kinase panels with DDX3X substrate to directly identify responsible kinases

  • Proximity Labeling Proteomics: BioID or APEX2 fusion to DDX3X to identify proximal kinases in cellular context

• Phosphatase Regulation Analysis:

  • Methodological Approach: Phosphatase inhibitor treatments to assess effects on T322 phosphorylation dynamics

  • Candidate Screening: Focus on phosphatases known to localize to stress granules or regulate RNA metabolism

  • Temporal Analysis: Measure dephosphorylation kinetics following stress removal

• Therapeutic Target Validation:

  • Conditional Models: Create conditional phosphomimetic (T322D/E) or phosphodeficient (T322A) DDX3X models

  • Phenotypic Rescue: Test if modulating kinase/phosphatase activity rescues cellular or organismal phenotypes

  • Small Molecule Screening: Develop high-throughput assays using the phospho-antibody to identify compounds that normalize DDX3X phosphorylation

• Pathway Integration Framework:

  • Stimulus-Response Mapping: Characterize which cellular stresses or signaling events trigger T322 phosphorylation

  • Signaling Cascade Elucidation: Establish the complete pathway from stimulus to kinase activation to T322 phosphorylation

  • Cross-regulation Analysis: Determine how T322 phosphorylation interacts with other DDX3X post-translational modifications

• Therapeutic Development Strategy:

  • Targeted Kinase Inhibition: Develop specific inhibitors for the identified kinase(s)

  • Phosphatase Activation: Explore phosphatase activators if hyperphosphorylation is pathogenic

  • Allosteric Modulators: Design compounds that stabilize either phosphorylated or non-phosphorylated conformations

  • RNA-Binding Modulators: Develop therapeutics that rescue RNA binding properties affected by phosphorylation

Understanding the regulatory mechanisms of T322 phosphorylation could provide critical insights for developing targeted therapies for DDX3X-associated disorders, particularly intellectual disability and potentially certain cancers where DDX3X function is dysregulated .

How do other post-translational modifications interact with T322 phosphorylation to regulate DDX3X function?

The interplay between T322 phosphorylation and other post-translational modifications (PTMs) on DDX3X represents a complex regulatory network worth investigating:

• Global PTM Profiling:

  • Mass Spectrometry Approach: Comprehensive PTM mapping of DDX3X under various cellular conditions

  • Temporal Analysis: Track changes in multiple modifications during stress responses or developmental processes

  • Site Interdependence: Determine if T322 phosphorylation affects the occurrence of other modifications

• Combinatorial Modification Analysis:

  • Multi-epitope Detection: Develop antibodies recognizing specific PTM combinations

  • Sequential Modification Timing: Establish order of modification events using pulse-chase experiments

  • Functional Consequences: Assess how different PTM combinations affect DDX3X:

    • RNA binding affinity

    • Protein-protein interactions

    • Subcellular localization

    • Enzymatic activity

• Crosstalk with Other Regulatory Mechanisms:

  • Methylation-Phosphorylation Interplay: Examine if arginine methylation affects T322 phosphorylation

  • Ubiquitination Dynamics: Determine if phosphorylation status alters ubiquitination and protein stability

  • SUMOylation Effects: Investigate potential crosstalk between SUMOylation and phosphorylation

• Structural Consequences Assessment:

  • Conformation Analysis: Use structural techniques (HDX-MS, FRET) to examine how PTM combinations alter protein conformation

  • Domain Interaction Studies: Determine if PTMs affect interactions between DDX3X domains

  • Molecular Dynamics Simulations: Model the effects of multiple PTMs on protein structure and dynamics

• Disease-Associated PTM Dysregulation:

  • Clinical Sample Analysis: Compare PTM patterns in patient-derived samples versus controls

  • Mutation Impact: Assess how disease-causing mutations affect the PTM landscape

  • PTM-Targeted Interventions: Develop strategies to normalize dysregulated PTM patterns

This multi-dimensional approach to understanding DDX3X regulation would provide unprecedented insights into how complex PTM patterns control this multifunctional protein in health and disease contexts. The relationship between T322 phosphorylation and other modifications may reveal novel therapeutic targets for DDX3X-associated disorders .