NCK1 Antibody

What is NCK1 and what are its primary functions in cellular signaling?

NCK1 is a cytoplasmic adaptor protein that functions primarily by connecting receptor tyrosine kinases to downstream effectors through its SH2 and SH3 domains. It plays essential roles in regulating the actin cytoskeleton, cell migration, and intracellular signal transduction.

Recent studies have demonstrated NCK1's involvement in multiple biological processes, including:

• Regulation of actin filament turnover in dendritic spines, affecting synapse formation and memory processing

• Mediation of inflammatory responses in endothelial cells during atherosclerosis development

• Promotion of cancer progression in various malignancies, including lung squamous cell carcinoma

The protein contains one SH2 domain and three SH3 domains that facilitate protein-protein interactions with phosphorylated tyrosine residues and proline-rich motifs, respectively. These structural features enable NCK1 to serve as a versatile scaffold in numerous signaling cascades related to cell growth, differentiation, and morphology.

How does NCK1 differ from its homolog NCK2?

Research has demonstrated that "only Nck1 deletion, but not Nck2 deletion, limited flow-induced NF-κB activation and proinflammatory gene expression" . In vivo studies confirmed that "Nck1-knockout mice showed reduced endothelial activation and inflammation in both models, disturbed flow– and high fat diet–induced atherosclerosis, whereas Nck2 deletion did not" .

These findings highlight the importance of distinguishing between these homologs in experimental design and interpretation, despite their structural similarities.

What are the structural domains of NCK1 and their functional significance?

NCK1 consists of several well-characterized protein interaction domains that mediate its adaptor functions:

• SH2 (Src Homology 2) Domain:

  • Located at the C-terminus

  • Binds to phosphorylated tyrosine residues on activated receptors and signaling proteins

  • Critical for disturbed flow-induced endothelial activation as identified through domain-swap experiments

• Three SH3 (Src Homology 3) Domains:

  • Positioned at the N-terminus

  • Bind to proline-rich motifs in target proteins

  • The first SH3 domain has been identified as critical for flow-induced endothelial activation

  • These domains facilitate interactions with various downstream effectors controlling actin dynamics

• Linker Regions:

  • Connect the functional domains

  • Provide structural flexibility and regulate interdomain interactions

The domain architecture enables NCK1 to function as a molecular scaffold that assembles multiple signaling partners, particularly in pathways regulating cytoskeletal reorganization and cellular responses to external stimuli.

How is NCK1 expression regulated in normal versus pathological states?

NCK1 expression undergoes significant regulation in various pathological conditions compared to normal states:

In cancer, particularly lung squamous cell carcinoma (LUSC), NCK1 shows upregulated expression. Research has revealed a specific regulatory mechanism where "NCK1-AS1 induced the upregulation of its nearby gene NCK adaptor protein 1 (NCK1) at the transcriptional level by interacting with the transcription factor MYC proto-oncogene (MYC)" . This suggests an oncogene-mediated transcriptional control mechanism.

In atherosclerosis, NCK1 expression in endothelial cells plays a critical role in disease progression. Studies found that "endothelial Nck1, but not hematopoietic Nck1, mediated this effect" , indicating tissue-specific regulation and function.

In neuronal systems, "NCK1 is expressed in postmitotic neurons but is dispensable for neuronal proliferation and migration in the developing hippocampus" , demonstrating developmental stage-specific regulation.

These findings collectively indicate that NCK1 expression is tightly controlled in a context-dependent manner, with dysregulation often associated with pathological conditions.

What are the optimal protocols for NCK1 detection using antibodies?

Based on published research protocols, here are the recommended methods for detecting NCK1 across different applications:

Western Blot Protocol:

• Sample preparation: Prepare cell or tissue lysates using appropriate lysis buffer

• Electrophoresis: Run samples on SDS-PAGE under reducing conditions

• Transfer: Transfer proteins to PVDF membrane

• Blocking: Block membrane with appropriate blocking buffer

• Primary antibody: Incubate with anti-NCK1 antibody at 1:500-1:2,000 dilution

• Washing: Wash membrane thoroughly

• Secondary antibody: Incubate with HRP-conjugated secondary antibody

• Detection: Visualize using enhanced chemiluminescence

• Expected result: NCK1 appears as a band at approximately 47 kDa

Immunocytochemistry Protocol:

• Cell fixation: Fix cells in paraformaldehyde

• Permeabilization: Permeabilize cell membranes (0.1-0.5% Triton X-100)

• Blocking: Block non-specific binding

• Primary antibody: Incubate with anti-NCK1 antibody at 1:50-1:200 dilution

• Secondary antibody: Apply fluorophore-conjugated secondary antibody

• Nuclear counterstaining: Counterstain with DAPI

• Expected result: Specific staining localized to cytoplasm

For detecting NCK1 in flow cytometry applications, the recommended dilution range is 1:50-1:100, while immunoprecipitation protocols typically use 1:10-1:50 dilution .

What controls should be included when working with NCK1 antibodies?

Proper experimental controls are essential for generating reliable data with NCK1 antibodies:

Positive Controls:

• Cell lines with confirmed NCK1 expression:

  • Human: HeLa, MCF-7, A172 glioblastoma, MOLT-4 leukemia cells

  • Mouse: NIH-3T3, BaF3 pro-B cells

  • Rat: Y3-Ag myeloid cells, kidney tissue

• Recombinant NCK1 protein can serve as a positive control for antibody validation

Negative Controls:

• NCK1 knockout or knockdown samples (siRNA or shRNA treated cells)

• Secondary antibody only controls to assess non-specific binding

• Blocking peptide controls to confirm specificity

Specificity Controls:

• Multiple antibodies targeting different NCK1 epitopes to confirm results

• Cross-reactivity assessment between NCK1 and the highly homologous NCK2

• Validation across multiple detection methods (Western blot, ICC, IP)

Loading and Processing Controls:

• For Western blots, include housekeeping proteins (β-actin, GAPDH, tubulin)

• For immunostaining, include counterstains to visualize cellular structure

• Process controls omitting primary antibody to assess secondary antibody specificity

These controls are critical for distinguishing genuine NCK1 signals from technical artifacts, particularly important when investigating subtle expression changes or novel interactions.

How can researchers troubleshoot non-specific binding with NCK1 antibodies?

Non-specific binding is a common challenge when working with antibodies. Here are strategies to improve NCK1 antibody specificity:

Optimization of Blocking Conditions:

• Use appropriate blocking agents (5% BSA, normal serum from secondary antibody species)

• Extend blocking time to ensure complete coverage of non-specific binding sites

• Consider adding 0.1-0.3% Triton X-100 to blocking solution for better penetration

Antibody Dilution Optimization:

• Test multiple dilutions, starting with manufacturer's recommendations (1:500-1:2,000 for WB, 1:50-1:200 for ICC/IHC)

• More dilute antibody solutions often reduce non-specific binding while maintaining specific signal

Washing Protocol Enhancement:

• Increase the number and duration of washing steps

• Use appropriate detergent concentration in wash buffers (0.05-0.1% Tween-20)

• Consider PBS with higher salt concentration for more stringent washing

Validation With Multiple Approaches:

• Use knockout/knockdown controls to confirm specificity

• Perform blocking peptide experiments to validate epitope specificity

• Compare results across multiple detection methods

Research has demonstrated successful NCK1 detection with specific cytoplasmic localization in MCF-7 cells , providing a reference for expected staining patterns when optimization is successful.

What are the key considerations when using NCK1 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (co-IP) is valuable for studying NCK1's protein interactions but requires specific considerations:

Antibody Selection:

• Choose antibodies validated for immunoprecipitation (recommended dilution 1:10-1:50)

• Consider antibodies targeting different epitopes to avoid interference with protein interactions

• Verify that the antibody doesn't disrupt the interaction of interest

Lysis Conditions:

• Use mild non-denaturing lysis buffers to preserve protein-protein interactions

• Include protease and phosphatase inhibitors to prevent degradation

• Optimize detergent type and concentration (typically 0.5-1% NP-40 or 0.5% Triton X-100)

• Adjust salt concentration to balance specificity with interaction preservation

Pre-clearing Step:

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Use appropriate control IgG matched to your primary antibody host species

Interaction Verification:

• Perform reciprocal co-IPs when possible (IP with partner antibody, detect NCK1)

• Include negative controls (unrelated proteins unlikely to interact with NCK1)

• Consider size-matched IgG controls to account for non-specific binding

Binding Partner Analysis:

• Research has identified IRAK-1 as a specific NCK1 binding partner

• When investigating novel interactions, consider validation through multiple methods

• Quantify interaction strength under different experimental conditions

How does NCK1 contribute to cancer progression mechanisms?

NCK1 has been implicated in multiple cancer progression mechanisms, with particularly strong evidence in lung squamous cell carcinoma (LUSC):

Oncogenic Activities in LUSC:
Research has demonstrated that "NCK1-AS1 prompted cell proliferation and migration, whilst impeded cell apoptosis in LUSC" . The long non-coding RNA NCK1-AS1 was found to induce upregulation of NCK1, suggesting a regulatory relationship critical for cancer progression.

Mechanistic Pathway:
"NCK1-AS1 induced the upregulation of its nearby gene NCK adaptor protein 1 (NCK1) at the transcriptional level by interacting with the transcription factor MYC proto-oncogene (MYC)" . This suggests a MYC-dependent regulatory mechanism for NCK1 expression in cancer.

Multi-Cancer Involvement:
"To date, NCK1 has been delineated to promote cancer development in several cancers, such as colorectal cancer and hepatocellular cancer" . This indicates that NCK1's oncogenic role extends beyond lung cancer.

Functional Effects:
NCK1 appears to enhance multiple hallmarks of cancer including:

• Increased cellular proliferation

• Enhanced cell migration and potential for metastasis

• Reduced apoptotic sensitivity

• Altered cellular signaling pathways

Rescue experiments in LUSC models confirmed that "NCK1 participated in the regulation of NCK1-AS1 on LUSC progression" , positioning NCK1 as a potential therapeutic target in oncology.

What is the role of NCK1 in neuronal development and memory formation?

NCK1 plays critical roles in neuronal development and memory formation through regulation of actin dynamics and dendritic spine formation:

Behavioral Phenotypes:
"Mice lacking NCK1 have impairments in both short-term and working memory, as well as spatial learning" . Interestingly, "female mice deficient in NCK1 fail at reversal learning in a spatial learning task" , suggesting sex-specific effects.

Neuronal Expression Pattern:
"NCK1 is expressed in postmitotic neurons but is dispensable for neuronal proliferation and migration in the developing hippocampus" . This indicates a specific role in mature neuronal function rather than early development.

Structural Effects:

Molecular Mechanism:
"The turnover of actin-filaments in dendritic spines is accelerated in neurons that lack NCK1" . This suggests that NCK1 functions to stabilize actin dynamics within dendritic spines, which is crucial for synapse formation and maintenance.

These findings collectively establish NCK1 as an important regulator of synaptic structure and function, with implications for understanding memory disorders and potential therapeutic interventions.

How does NCK1 participate in inflammatory signaling pathways?

NCK1 plays a selective and critical role in inflammatory signaling pathways, particularly in the context of endothelial activation and atherosclerosis:

Inflammatory Pathway Regulation:
"Only Nck1 deletion, but not Nck2 deletion, limited flow-induced NF-κB activation and proinflammatory gene expression" . This indicates a specific role for NCK1 in regulating the key inflammatory transcription factor NF-κB.

In Vivo Inflammatory Effects:
"Nck1-knockout mice showed reduced endothelial activation and inflammation in both models, disturbed flow– and high fat diet–induced atherosclerosis, whereas Nck2 deletion did not" . This demonstrates the importance of NCK1 in inflammatory processes related to atherosclerosis.

Proinflammatory Gene Regulation:
"Oscillatory flow–induced NF-κB activation (p65 Ser536 phosphorylation) and VCAM-1/ICAM-1 protein expression and mRNA levels were blunted by Nck1 siRNA" . These adhesion molecules are critical for leukocyte recruitment during inflammation.

IRAK-1 Interaction:
A key mechanistic finding was "identifying interleukin 1 type I receptor kinase-1 (IRAK-1) as a Nck1-selective binding partner, demonstrating that IRAK-1 activation by disturbed flow required Nck1 in vitro and in vivo" . This links NCK1 directly to IL-1 signaling, a major proinflammatory pathway.

Systemic Inflammatory Markers:
"Nck1-KO mice showed significant reductions in the plasma levels of several proinflammatory mediators, including interleukin 1α (IL-1α), IL-1β, TNF-α, and MCP-1" , indicating broad effects on inflammatory processes.

These findings position NCK1 as a potential therapeutic target for inflammatory conditions, particularly atherosclerosis and vascular inflammation.

What domain-specific functions have been identified for NCK1?

Understanding the functions of specific NCK1 domains provides critical insights into its molecular mechanisms:

SH2 Domain Functions:

• "Domain-swap experiments and point mutations identified the Nck1 SH2 domain...as critical for flow-induced endothelial activation"

• This C-terminal domain mediates interactions with phosphorylated tyrosine residues

• Essential for NCK1's role in inflammatory signaling pathways

• Distinguishes NCK1 functionality from NCK2 despite structural similarity

First SH3 Domain Functions:

• "The first SH3 domain has been identified as critical for flow-induced endothelial activation"

• Mediates interaction with proline-rich motifs in partner proteins

• Contributes to NCK1's specificity in signaling pathways

• Key determinant in differentiating NCK1 from NCK2 functionality

Domain Cooperation:
The coordinated action of multiple NCK1 domains appears necessary for its biological functions. Research suggests that both the SH2 domain and SH3 domains work together to orchestrate proper protein-protein interactions in various cellular contexts.

These domain-specific insights provide valuable direction for designing targeted interventions that could modulate specific NCK1 functions while preserving others.

How can researchers distinguish between NCK1 and NCK2 in experimental systems?

Distinguishing between the highly homologous NCK1 and NCK2 proteins presents significant challenges but is essential for accurate experimental interpretation:

Antibody-Based Discrimination:

• Select antibodies validated for specific detection with no cross-reactivity

• Verify antibody specificity using knockout/knockdown controls

• "Nck1- and Nck2-selective siRNAs that result in a 75% and 85% knockdown, respectively, without affecting the expression of the other isoform" demonstrate the feasibility of selective targeting

Functional Validation:

• Use functional assays that capitalize on known differential activities

• "Nck1-depleted cells showed significantly less NF-κB p65 phosphorylation and nuclear translocation, whereas Nck2 depletion did not affect NF-κB activation by flow"

• "Oscillatory flow–induced NF-κB activation and VCAM-1/ICAM-1 protein expression and mRNA levels were blunted by Nck1 siRNA, whereas Nck2 depletion had no significant effects"

Genetic Approaches:

• Use isoform-specific knockout models

• "MAECs isolated from Nck1-KO mice showed...remarkable reduction in NF-κB activation following shear stress, whereas MAECs from iEC-Nck2–KO mice showed the usual shear stress–induced NF-κB activation"

• Create cell lines with tagged versions of each protein to facilitate discrimination

Interaction Partner Analysis:

• Focus on specific binding partners unique to each isoform

• "IRAK-1 as a Nck1-selective binding partner" provides a means to specifically study NCK1-mediated processes

These approaches enable researchers to confidently distinguish between these highly similar proteins and accurately attribute specific functions to each isoform.

What factors might affect variability in NCK1 detection between experiments?

Several factors can contribute to variability in NCK1 detection and expression levels between experiments:

Biological Variables:

• Cell cycle stage: Expression levels may fluctuate during different phases

• Cell density and confluency: Contact inhibition may alter signaling pathways

• Passage number: Cellular phenotypes can drift with extended culture

• Growth factor variations in media: Serum components may influence expression

• Cellular stress responses: Heat shock, oxidative stress, or nutrient deprivation

Technical Variables:

• Sample preparation: Variations in lysis buffer composition or extraction efficiency

• Antibody performance: Lot-to-lot variability in antibody reactivity

• Detection method sensitivity: Western blot vs. immunofluorescence vs. flow cytometry

• Fixation conditions: Different fixatives can affect epitope accessibility

• Permeabilization efficiency: Incomplete permeabilization may reduce detection

Experimental Design Factors:

• Treatment timing: Variations in exposure time to experimental conditions

• Environmental factors: Temperature, CO2 levels, humidity in cell culture

• Protocol consistency: Subtle variations in experimental procedures

Control Strategies:

• Include consistent positive controls across experiments

• Use multiple detection methods for validation

• Standardize protocols with detailed SOPs

• Run multiple biological and technical replicates

• Consider internal normalization to account for loading variations

Understanding these variables and implementing appropriate controls allows researchers to generate more consistent and reliable NCK1 data across experiments.

How can researchers resolve discrepancies in NCK1 functional studies?

When faced with discrepancies in NCK1 functional studies, several approaches can help resolve inconsistencies:

Comprehensive Loss-of-Function Studies:

• Use multiple knockdown approaches (siRNA, shRNA) with different target sequences

• Apply CRISPR/Cas9 gene editing for complete knockout

• Use conditional knockout models to study tissue-specific effects

• Perform rescue experiments with wild-type NCK1 to confirm specificity

Domain-Specific Analysis:

• Utilize domain mutants to identify specific functional regions

• "Domain-swap experiments and point mutations identified the Nck1 SH2 domain and the first SH3 domain as critical for flow-induced endothelial activation"

• Create chimeric proteins (e.g., NCK1/NCK2 domain swaps) to assess domain-specific functions

Context-Dependent Investigations:

• Study NCK1 function across multiple cell types

• Assess function under various stimulation conditions

• Consider temporal dynamics of NCK1-dependent responses

• Examine potential compensatory mechanisms (e.g., NCK2 upregulation in NCK1-deficient systems)

Interaction Network Analysis:

• Identify and validate key binding partners in your experimental system

• "IRAK-1 as a Nck1-selective binding partner" demonstrates the importance of specific interactions

• Use co-immunoprecipitation and mass spectrometry to identify context-specific interactors

• Disrupt specific interactions through targeted mutations

These systematic approaches can help resolve apparent contradictions in NCK1 functional studies and develop a more nuanced understanding of its context-dependent roles.

What are the emerging research directions for NCK1 antibody applications?

Several promising research directions are emerging for NCK1 antibody applications:

Therapeutic Target Validation:

• Using antibodies to validate NCK1 as a potential drug target in cancer

• "NCK1-AS1 prompted cell proliferation and migration, whilst impeded cell apoptosis in LUSC" suggests oncological applications

• Evaluating NCK1 inhibition in atherosclerosis based on findings that "Nck1-knockout mice showed reduced endothelial activation and inflammation"

Domain-Specific Antibodies:

• Developing antibodies that target specific functional domains of NCK1

• Creating tools that distinguish between active and inactive conformations

• Engineering antibodies that selectively block specific protein-protein interactions

Post-Translational Modification Mapping:

• Generating modification-specific antibodies (phospho-specific, etc.)

• Tracking dynamic changes in NCK1 modifications during signaling events

• Correlating modifications with functional outcomes

Advanced Imaging Applications:

• Using NCK1 antibodies for super-resolution microscopy

• Developing tools for live-cell imaging of NCK1 dynamics

• Creating proximity ligation assays to study NCK1 interaction networks in situ

Diagnostic Applications:

• Exploring NCK1 as a potential biomarker in cancer and inflammatory diseases

• Developing antibody-based diagnostic tests for NCK1 expression levels

• Correlating NCK1 expression patterns with disease progression and outcomes

These emerging directions highlight the continuing importance of NCK1 antibodies as tools for both basic research and translational applications across multiple disease areas.

Future perspectives on NCK1 research applications

The future of NCK1 research holds promising directions across multiple fields. In cancer biology, the emerging understanding of NCK1's role in promoting cell proliferation and migration positions it as a potential therapeutic target. Developing specific inhibitors of NCK1 function or its regulatory pathways may provide novel anti-cancer strategies.

In cardiovascular medicine, NCK1's selective role in atherogenic inflammation suggests possibilities for targeted interventions that could reduce inflammatory vascular disease without compromising other immune functions. The identification of IRAK-1 as a specific NCK1 binding partner provides a potential mechanism for such selective targeting.

In neuroscience, NCK1's involvement in dendritic spine formation and memory processing opens avenues for understanding and potentially addressing cognitive disorders. The sex-specific differences observed in NCK1-deficient mice further highlight the importance of considering sex as a biological variable in NCK1 research.

Methodologically, advances in antibody engineering, CRISPR gene editing, and imaging technologies will continue to enhance our ability to study NCK1 with greater precision. These technical developments will likely reveal more nuanced understanding of NCK1's context-dependent functions and regulatory mechanisms.