DOK1 Antibody

What is DOK1 and what are its primary cellular functions?

DOK1 is a multi-domain adapter protein that acts as a negative regulator of signaling pathways involved in several cellular functions. It functions downstream of various receptor and nonreceptor tyrosine kinase cascades and plays a key role in regulating cellular activities. Specifically, DOK1 inhibits cell proliferation, down-regulates MAP kinase activity, and has an opposing role in leukemogenesis. It also promotes cell spreading, motility, and apoptosis under certain conditions .

DOK1 has been identified as a tumor suppressor gene whose expression is altered and down-regulated in chronic lymphocytic leukemia and other malignancies. Its tumor-suppressive properties suggest that genetic alterations or low expression of DOK1 and its related member DOK2 may be involved in various hematopoietic and nonhematopoietic malignancies .

What applications can DOK1 antibodies be used for in research?

DOK1 antibodies can be employed in multiple experimental applications:

The antibody has been tested with human samples and shows positive Western blot detection in K-562 cells and immunohistochemistry detection in human ovary cancer tissue . For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 can be used as an alternative .

What is the subcellular localization of DOK1 protein?

DOK1 exhibits a complex subcellular distribution pattern. While it is predominantly localized in the cytoplasm and plasma membrane region, a small proportion can also be found in the nucleus in some cells . Interestingly, DOK1's nuclear-cytoplasmic trafficking is regulated by an active nuclear export mechanism. When cells are treated with Leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export, over 90% of endogenous DOK1 accumulates in the nucleus .

This nuclear accumulation occurs rapidly within 10 minutes of LMB treatment and gradually increases to reach a peak around 1 hour and 30 minutes, suggesting that DOK1 is actively exported from the nucleus through a CRM1/exportin pathway and likely contains a functional Nuclear Export Signal (NES) .

How does DOK1 phosphorylation affect its function in cell signaling?

DOK1 undergoes complex phosphorylation patterns that significantly impact its function. While tyrosine phosphorylation of DOK1 has been well-documented, recent research has identified crucial serine phosphorylation events mediated by IKKβ (IκB kinase β). IKKβ physically associates with and phosphorylates DOK1 at specific serine residues—S439, S443, S446, and S450 .

These phosphorylation events occur in response to various stimuli including TNF-α, IL-1, and γ-radiation. Functionally, these serine phosphorylation sites are critical for DOK1's ability to inhibit platelet-derived growth factor (PDGF)-induced ERK1/2 phosphorylation and cell growth. When these serines are mutated to alanines (preventing phosphorylation), DOK1 loses its ability to inhibit cell growth and PDGF-induced ERK1/2 activation .

Moreover, serine phosphorylation is essential for DOK1's promotion of cell motility. Mutation of these serines to glutamic acid (mimicking phosphorylation) further enhances DOK1's ability to promote cell motility, underscoring the importance of these post-translational modifications .

What is the relationship between DOK1/DOK2 and T cell immunity?

DOK1 and DOK2 play significant regulatory roles in T cell signaling and memory formation. Research has shown that depletion of both DOK1 and DOK2 in CD8+ T cells after in vitro pre-stimulation leads to:

• A higher percentage of effector memory T cells

• Upregulation of TCR signaling cascade induced by CD3 monoclonal antibodies

• Increased levels of phosphorylated AKT and ERK, two major phosphoproteins involved in T cell functions

Interestingly, this enhanced TCR signaling was only observed in pre-stimulated CD8+ T cells but not in naïve CD8+ T cells. Despite the improved TCR signaling shown upon stimulation via CD3 monoclonal antibodies, pre-stimulated Dok1/Dok2 double knockout CD8+ T cells did not demonstrate any increase in their activation or cytotoxic capacities against melanoma cell lines expressing hgp100 in vitro .

These findings suggest that while DOK1 and DOK2 regulate aspects of T cell memory formation and signaling, their role in cytotoxic functions against cancer cells may be more complex and context-dependent.

How does nuclear-cytoplasmic shuttling affect DOK1 function?

DOK1's ability to shuttle between the cytoplasm and nucleus adds another layer of complexity to its regulatory functions. Studies using fluorescence microscopy and nuclear export inhibitors have revealed that DOK1 contains functional nuclear export signals and can actively transit through the nucleus .

Different domains of DOK1 exhibit distinct localization patterns:

• The PH domain (amino acids 1-150) shows diffuse staining with strong accumulation in the plasma membrane due to association with phospholipids

• Amino acids 250-430 demonstrate cytoplasmic localization that shifts to nuclear accumulation when nuclear export is inhibited

• Other regions (150-250 and 430-481) show diffuse distribution throughout both cytoplasm and nucleus

This nuclear-cytoplasmic shuttling capability suggests that DOK1 may have distinct functions in different cellular compartments. For example, nuclear DOK1 might interact with transcription factors or chromatin-associated proteins, whereas cytoplasmic DOK1 primarily functions in signal transduction pathways near the plasma membrane. This compartmentalization adds another regulatory dimension to DOK1's function that researchers should consider when designing experiments and interpreting results.

What are the optimal conditions for detecting DOK1 using Western blot?

For optimal Western blot detection of DOK1, researchers should consider the following protocol:

• Sample preparation:

  • Use cells known to express DOK1 (e.g., K-562 cells show positive results)

  • Extract proteins using a buffer containing phosphatase inhibitors if studying phosphorylation states

  • Denature samples in loading buffer containing SDS and β-mercaptoethanol

• Gel electrophoresis and transfer:

  • Use 10% SDS-PAGE gels for optimal resolution of DOK1 (observed molecular weight: 62 kDa)

  • Transfer to PVDF or nitrocellulose membranes using standard wet or semi-dry transfer methods

• Antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST

  • Dilute primary DOK1 antibody 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST

  • Incubate with peroxidase-conjugated secondary antibody (e.g., anti-rabbit IgG) for 1 hour at room temperature

• Detection:

  • Visualize using enhanced chemiluminescence (ECL) substrates like SuperSignal West Pico or SuperSignal West Femto

  • Expected band size for DOK1 is approximately 62 kDa

For phospho-specific detection of DOK1, phospho-specific antibodies against sites such as pS443 and pS450 can be used following a similar protocol, but blocking and antibody dilution should be performed in 5% BSA rather than milk to avoid interference with phospho-epitopes .

How should immunohistochemistry for DOK1 be optimized?

For successful immunohistochemical detection of DOK1 in tissue samples:

• Tissue preparation:

  • Fix tissue samples in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-6 μm thickness

  • Mount sections on positively charged slides

• Antigen retrieval:

  • Primary recommendation: Use TE buffer pH 9.0 for antigen retrieval

  • Alternative method: Citrate buffer pH 6.0 can also be effective

  • Heat-induced epitope retrieval methods (pressure cooker, microwave, or water bath) are recommended

• Immunostaining procedure:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with serum-free protein block

  • Dilute primary DOK1 antibody 1:50-1:500 depending on tissue type and detection system

  • Incubate at 4°C overnight or at room temperature for 1-2 hours

  • Apply appropriate detection system (HRP-polymer or secondary antibody)

  • Develop with DAB and counterstain with hematoxylin

• Validation controls:

  • Include positive control tissue (human ovary cancer tissue has shown positive results)

  • Include negative controls by omitting primary antibody

  • If possible, include tissue from DOK1 knockout models as specificity controls

Optimization may require testing different antibody dilutions and antigen retrieval methods for each specific tissue type under investigation.

What approaches can be used to study DOK1 phosphorylation?

Studying DOK1 phosphorylation requires specialized techniques:

• Phospho-specific Western blotting:

  • Use phospho-specific antibodies targeting known phosphorylation sites (e.g., pS443, pS446, pS450)

  • Include appropriate controls:

    • Wild-type DOK1 vs. phosphorylation site mutants (e.g., S to A mutations)

    • Treatment with phosphatase inhibitors vs. without

    • Stimulation with known inducers of phosphorylation (TNF-α, IL-1, γ-radiation)

• Immunoprecipitation coupled with phospho-detection:

  • Immunoprecipitate DOK1 using specific antibodies

  • Probe with phospho-specific antibodies or general phospho-serine/phospho-tyrosine antibodies

  • Alternatively, analyze immunoprecipitates by mass spectrometry to identify phosphorylation sites

• In vitro kinase assays:

  • Use recombinant IKKβ or other kinases with purified DOK1 or DOK1 fragments

  • Include [γ-³²P]ATP to detect phosphorylation

  • Analyze by autoradiography or phosphorimaging

• Functional validation using phospho-mutants:

  • Generate DOK1 constructs with serine-to-alanine mutations to prevent phosphorylation

  • Generate DOK1 constructs with serine-to-glutamate mutations to mimic phosphorylation

  • Compare the functional effects of these mutants on cell growth, ERK activation, and cell motility

What are common issues in DOK1 detection and how can they be resolved?

Researchers may encounter several challenges when detecting DOK1:

• Weak or no signal in Western blot:

  • Ensure sample contains DOK1 (K-562 cells are known to express DOK1)

  • Optimize antibody dilution (try 1:500 instead of 1:2000)

  • Increase protein loading amount

  • Extend exposure time

  • Check antibody storage conditions (store at -20°C, avoid repeated freeze-thaw cycles)

  • Use enhanced chemiluminescence detection systems for greater sensitivity

• Multiple bands or unexpected band size:

  • DOK1's calculated molecular weight is 52 kDa, but observed molecular weight is typically 62 kDa

  • Post-translational modifications may affect migration

  • Phosphorylation states can create band shifts

  • Verify specificity using DOK1 knockout or knockdown controls

  • Try different sample preparation methods to reduce protein degradation

• Poor or inconsistent IHC staining:

  • Optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Adjust antibody concentration (recommended range: 1:50-1:500)

  • Increase incubation time or temperature

  • Ensure tissue fixation is optimal (overfixation can mask epitopes)

  • Test different detection systems (polymer-based systems often provide higher sensitivity)

• Nuclear vs. cytoplasmic localization discrepancies:

  • DOK1 shuttles between nucleus and cytoplasm

  • Different fixation methods may preserve different localization patterns

  • Treatment with LMB can be used as a positive control for nuclear accumulation

  • Consider using subcellular fractionation followed by Western blot as a complementary approach

How can contradictory results between DOK1 expression and function be reconciled?

Researchers sometimes encounter contradictory results when studying DOK1. Here are strategies to reconcile such discrepancies:

• Cell type considerations:

  • DOK1 function may be cell-type specific

  • In T cells, DOK1/DOK2 effects differ between naïve and pre-stimulated cells

  • Compare your results with published data in the same cell type

• Context-dependent phosphorylation:

  • DOK1 undergoes both tyrosine and serine phosphorylation

  • Different stimuli may induce distinct phosphorylation patterns

  • Specific phosphorylation sites (S439, S443, S446, S450) are critical for function

  • Use phospho-specific antibodies or phospho-mutants to distinguish different phosphorylation states

• Nuclear-cytoplasmic distribution:

  • DOK1 function may differ based on subcellular localization

  • Nuclear export inhibition with LMB dramatically alters DOK1 localization

  • Examine both total expression and subcellular distribution

• Functional redundancy with other DOK family members:

  • DOK1 and DOK2 may have overlapping functions

  • Consider double knockouts/knockdowns to reveal phenotypes masked by redundancy

  • Analyze expression of multiple DOK family members simultaneously

• Experimental design considerations:

  • Acute vs. chronic manipulation may yield different results

  • Overexpression vs. endogenous studies might show different effects

  • In vitro vs. in vivo contexts may reveal different aspects of DOK1 function

How should DOK1 phosphorylation be analyzed in the context of signaling pathways?

When analyzing DOK1 phosphorylation within signaling pathways:

• Temporal dynamics assessment:

  • Perform time-course experiments after stimulation

  • DOK1 nuclear accumulation peaks around 1.5 hours after LMB treatment

  • Compare DOK1 phosphorylation kinetics with other signaling events

• Pathway integration analysis:

  • Simultaneously monitor DOK1 phosphorylation and downstream effectors (e.g., ERK1/2, AKT)

  • Wild-type DOK1 inhibits PDGF-induced ERK1/2 phosphorylation, while phospho-site mutants (A439, A443, A446, A450) do not

  • Use pathway inhibitors to establish causal relationships

• Multi-modification analysis:

  • Consider interactions between different phosphorylation sites

  • Analyze how tyrosine and serine phosphorylation might influence each other

  • Use phospho-mimetic and phospho-deficient mutants to dissect the contributions of specific sites

• Stimulus-specific regulation:

  • Different stimuli (TNF-α, IL-1, γ-radiation) can induce DOK1 phosphorylation

  • Compare phosphorylation patterns across different stimuli

  • Use stimulus-specific pathway inhibitors to identify responsible kinases

• Functional correlation:

  • Link specific phosphorylation events to functional outcomes

  • DOK1 serine phosphorylation affects:

    • Inhibition of cell proliferation

    • Regulation of PDGF-induced ERK1/2 activation

    • Promotion of cell motility

  • Use mutational analysis to establish causality between phosphorylation and function