DOK3 Antibody

What is DOK3 and what are its primary functions in the immune system?

DOK3 is an enzymatically inert adaptor/scaffolding protein that provides a docking platform for assembling multimolecular signaling complexes. It functions primarily as:

• A negative regulator of JNK signaling in B-cells through interaction with SHIP (SH2-containing inositol phosphatase)

• A critical regulator of immune responses, playing key roles in cell signaling pathways that control inflammation and immune cell activation

• A modulator of ABL1 function

• An essential component for optimal T-cell-dependent antibody responses

DOK3 contains a pleckstrin homology region (amino acids 63-179) and an IRS-type phosphotyrosine binding domain (PTB) (amino acids 213-317), which facilitate its interactions with various signaling molecules . When tyrosine residues in DOK3 are phosphorylated, the protein can interact with SH2 and SH3 containing proteins, leading to inhibition of signaling from immunoreceptors .

How is DOK3 involved in plasma cell differentiation?

DOK3 plays a crucial role in the differentiation of antigen-specific plasma cells (PCs). Research using DOK3-deficient mice has revealed:

• Despite having expanded germinal center (GC) B cell populations, DOK3-deficient mice exhibit significantly impaired antigen-specific antibody responses

• DOK3 deficiency does not affect the generation of antigen-specific GC B cells but severely compromises the differentiation of these cells into antibody-secreting plasma cells

• DOK3-deficient mice generate significantly fewer NP-specific IgG1 plasma cells in both spleen and bone marrow compared to wild-type mice

• The pool of long-lived plasma cells in bone marrow remains drastically reduced in DOK3-deficient mice even 28 days after immunization

These findings indicate that DOK3 signaling is required for the efficient transition of activated B cells into plasma cells, which is essential for optimal humoral immune responses.

What applications are DOK3 antibodies validated for in research settings?

DOK3 antibodies have been validated for multiple experimental applications:

For optimal results, each laboratory should determine the appropriate dilution for their specific experimental conditions . DOK3 antibodies have demonstrated reactivity with both human and mouse samples , making them suitable for comparative studies across species.

What is the best approach for detecting DOK3 by Western blot?

For optimal detection of DOK3 by Western blot:

• Sample preparation: Prepare lysates from relevant tissues or cells (e.g., B cells, macrophages, or peripheral blood mononuclear cells)

• Antibody selection: Choose an antibody with validated WB application (monoclonal or polyclonal based on experimental needs)

• Running conditions: Use reducing conditions and appropriate buffer systems (e.g., Immunoblot Buffer Group 2)

• Antibody dilution: Start with 0.25 μg/mL for monoclonal antibodies or 1:500-1:2000 for polyclonal antibodies

• Secondary antibody: Use an appropriate HRP-conjugated secondary antibody (e.g., Anti-Mouse IgG for monoclonal or Anti-Rabbit IgG for polyclonal primary antibodies)

• Expected band size: Look for a specific band at approximately 53-65 kDa

It's important to note that while the predicted molecular weight of DOK3 is around 53 kDa , it may appear at approximately 65 kDa on Western blots due to post-translational modifications .

Why might DOK3 antibodies show different banding patterns across cell types?

Different banding patterns when detecting DOK3 can result from:

• Alternative splicing: Human DOK3 has four known isoforms produced by alternative splicing, with protein lengths of 496, 330, 228, and 216 amino acids . The isoforms have different N-terminal and C-terminal truncations and substitutions.

• Post-translational modifications: DOK3 undergoes tyrosine phosphorylation upon immunoreceptor-mediated cellular stimulation , which can alter its apparent molecular weight.

• Cell type-specific expression: DOK3 expression is particularly elevated in B cells and macrophages , and different cell types may express different isoforms at varying levels.

• Protein interactions: When DOK3 is phosphorylated, it interacts with proteins such as SHIP and CSK via their SH2 domains , which could affect antibody recognition or protein migration.

To address these variations, researchers should:

• Include appropriate positive controls (e.g., PBMC lysates)

• Consider using multiple antibodies targeting different epitopes

• Compare results across different experimental methods

How can I optimize storage and handling of DOK3 antibodies to maintain reactivity?

To maintain optimal reactivity of DOK3 antibodies:

Storage recommendations:

• Store unopened antibodies at -20 to -70°C for up to 12 months from date of receipt

• After reconstitution, store at 2-8°C under sterile conditions for up to 1 month

• For longer storage after reconstitution, aliquot and store at -20 to -70°C for up to 6 months

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

Buffer considerations:

• Many DOK3 antibodies are supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• This formulation helps maintain antibody stability during storage

Handling practices:

• Centrifuge the vial briefly before opening

• Prepare working dilutions on the day of use

• Return to storage conditions immediately after use

How does DOK3 deficiency impact the germinal center reaction and what are the implications for studying antibody-mediated immunity?

DOK3 deficiency creates an interesting paradox in germinal center (GC) reactions and antibody-mediated immunity:

These findings suggest that DOK3 functions as a critical checkpoint regulator in the GC reaction, specifically in the terminal differentiation of GC B cells into plasma cells. Researchers studying antibody-mediated immunity should consider:

• Using DOK3 deficiency models to specifically investigate plasma cell differentiation mechanisms

• Examining the molecular pathways linking DOK3 to transcription factors essential for plasma cell differentiation

• Investigating potential compensation mechanisms that allow normal GC formation but impair plasma cell development

What molecular mechanisms underlie DOK3's role in signaling regulation, and how can researchers experimentally probe these pathways?

DOK3 regulates signaling through several molecular mechanisms:

• Phosphotyrosine-dependent interactions:

  • Upon tyrosine phosphorylation, DOK3 interacts with SHIP and CSK via their SH2 domains

  • Both phosphorylated Y381 and Y398 are required for interaction with SHIP, while only pY381 is needed for CSK interaction

• Pleckstrin homology and PTB domain functions:

  • DOK3 contains a pleckstrin homology region (aa 63-179) and an IRS-type phosphotyrosine binding domain (aa 213-317)

  • The PTB domain mediates binding to ABL in a kinase-dependent manner

• Negative regulation of signaling pathways:

  • DOK3 serves as a negative regulator of JNK signaling in B-cells through interaction with SHIP

  • It acts as a signaling hub for recruiting inhibitory molecules in immune cells

Researchers can experimentally probe these pathways using:

Experimental approaches:

• Site-directed mutagenesis: Generate Y381F and Y398F mutants to disrupt specific interactions

• Domain deletion studies: Create constructs lacking the PH or PTB domains

• Proximity labeling: Use BioID or APEX2 fusions to identify novel interacting partners

• Phosphoproteomic analysis: Compare signaling networks in wild-type versus DOK3-deficient cells

• Live-cell imaging: Monitor DOK3 localization during cell activation using fluorescently tagged constructs

Controls and validation:

• Verify DOK3 expression and phosphorylation status using validated antibodies

• Include both positive controls (stimulated B cells) and negative controls (non-lymphoid cells with low DOK3 expression)

How might DOK3 function as a therapeutic target in inflammatory or autoimmune diseases?

Given DOK3's role as a negative regulator of immune signaling, it represents a potential therapeutic target:

• Rationale for targeting DOK3:

  • DOK3 negatively regulates JNK signaling in B-cells

  • It modulates immunoreceptor-mediated cellular stimulation

  • DOK3 is involved in controlling inflammation and immune cell activation

• Potential therapeutic approaches:

  • Enhancement strategies: Augmenting DOK3 function could dampen excessive immune activation in autoimmune diseases

  • Inhibition strategies: In specific contexts where enhanced antibody responses are desired (e.g., vaccination), temporary DOK3 inhibition might increase plasma cell generation

• Disease relevance:

  • Autoimmune disorders where B cell hyperactivity is implicated

  • Chronic inflammatory conditions

  • Cancer immunotherapy, where modulating immune responses is critical

Future research directions should explore:

• Development of small molecules or peptides that can modulate DOK3 phosphorylation or protein interactions

• Cell-type specific targeting approaches to limit off-target effects

• Temporal regulation of DOK3 function to fine-tune immune responses

What are the technical considerations when using DOK3 antibodies in multiplex immunofluorescence studies?

When incorporating DOK3 antibodies into multiplex immunofluorescence studies:

• Antibody selection considerations:

  • Choose antibodies from different host species to avoid cross-reactivity

  • Verify that the DOK3 antibody has been validated for immunofluorescence applications

  • Consider using monoclonal antibodies when possible for consistent epitope recognition

• Optimization parameters:

  • Dilution: Start with recommended dilutions (1:200-1:1000 for IF) and optimize

  • Antigen retrieval: Test different methods (heat-induced vs. enzymatic)

  • Blocking: Use appropriate blocking reagents to minimize background

  • Detection system: Select fluorophores with minimal spectral overlap

• Controls and validation:

  • Include single-stained controls to assess bleed-through

  • Use known DOK3-expressing tissues (e.g., tonsil, lymph nodes) as positive controls

  • Include isotype controls to assess non-specific binding

• Technical implementation:

  • Consider the order of antibody application (typically start with the lowest concentration)

  • Allow sufficient washing between steps to reduce background

  • Use appropriate mounting media with anti-fade properties for signal preservation

By carefully optimizing these parameters, researchers can successfully incorporate DOK3 detection into multiplex immunofluorescence panels for comprehensive analysis of immune cell signaling networks.