NCKAP1 Antibody

What is NCKAP1 and why is it important for cellular function?

NCKAP1 (NCK-associated protein 1), also known as NAP125 or membrane-associated protein HEM-2, is a 1,128 amino acid single-pass membrane protein belonging to the HEM-1/HEM-2 family. It exists in two alternatively spliced isoforms and plays a crucial role in actin cytoskeleton regulation . NCKAP1 is widely expressed throughout the body but shows highest expression in heart, brain, and skeletal muscle tissues .

The protein's primary function involves regulating Rac-dependent actin remodeling as part of a lamellipodial complex with WAVE2, Abi-1, and CYFIP1 . This makes NCKAP1 essential for cellular processes that require cytoskeletal reorganization, including cell migration, adhesion, and morphogenesis. Notably, NCKAP1 localizes to the cytoplasmic side of the lamellipodium membrane and is encoded by a gene mapping to human chromosome 2q32.1 .

Validation of NCKAP1 antibodies should follow a multi-step approach:

• Positive and negative control tissues: Brain and heart tissues (high expression) serve as positive controls, while tissues with minimal NCKAP1 expression can serve as negative controls .

• Knockdown/knockout verification: Using siRNA or CRISPR to reduce or eliminate NCKAP1 expression creates essential negative controls to confirm antibody specificity .

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of NCKAP1 helps confirm detection specificity .

• Recombinant protein standards: Including purified NCKAP1 protein as a positive control in Western blots helps establish the correct molecular weight (approximately 125 kDa) .

• Cross-reactivity assessment: Testing the antibody against related proteins, particularly NCKAP1L (the hematopoietic homolog), to ensure specificity .

These validation steps are critical because of the potential for cross-reactivity and the existence of multiple isoforms of NCKAP1 .

How does NCKAP1 expression correlate with cancer progression and metastasis?

NCKAP1 exhibits context-dependent roles in cancer progression that vary by cancer type:

In colorectal cancer (CRC), NCKAP1 appears to promote metastasis. Research shows significantly increased NCKAP1 in blood RNA of patient-derived xenograft models of colon cancer . Knockdown of NCKAP1 in human colon cancer cell lines (HCT116 and HT29) resulted in:

• Reduced wound healing capacity

• Inhibition of migration and invasion

• Enhanced adherent cell junctions (increased CDH1 expression)

• Strengthened cytoskeleton (increased phalloidin expression)

• In metastasis xenograft models, significant reduction in tumor growth and liver metastasis

Contrastingly, in hepatocellular carcinoma (HCC), NCKAP1 functions as a tumor suppressor. Analysis showed:

• Correlation with favorable patient outcomes

• Inhibition of cell growth in vitro and in vivo models

• Prevention of cell cycle progression into G2/M phase

• Upregulation of tumor suppressors Rb1 and p53

This dichotomous role highlights the importance of tissue context when studying NCKAP1 function in cancer. The contrasting functions may relate to differential expression of NCKAP1's interacting partners (such as WASF1) across cancer types .

What methodological considerations are critical when designing experiments to study NCKAP1's role in epithelial-mesenchymal transition (EMT)?

When investigating NCKAP1's role in EMT, researchers should consider:

• Induction models: TGFβ1 treatment of HCT116 cells has been validated as an effective model for inducing a mesenchymal state. In this model, NCKAP1 knockdown inhibits migration and alters key EMT markers (increased CTNNB1, decreased FN expression) .

• Marker selection: A comprehensive panel of markers should include:

  • Epithelial markers: E-cadherin (CDH1), β-catenin (CTNNB1)

  • Mesenchymal markers: Fibronectin (FN), N-cadherin

  • Cytoskeletal markers: Phalloidin (F-actin)

  • Transcription factors: SNAI1, TWIST, ZEB1/2

• Temporal considerations: EMT is a dynamic process, requiring time-course analysis rather than single time-point measurements.

• 3D culture models: These more accurately recapitulate in vivo cell behaviors compared to traditional 2D cultures.

• In vivo validation: Two complementary metastasis models have been established:

  • Intrasplenic injection of shRNA-NCKAP1 transfected cells

  • Cecal pouch implantation of tumor tissue generated from knockdown cells

Both models demonstrated NCKAP1's impact on metastatic potential, with markedly reduced liver metastasis when NCKAP1 was suppressed .

How should researchers address potential contradictions in NCKAP1 function between different disease models?

The contradictory roles of NCKAP1 across disease models require careful experimental design:

A rigorous approach would include parallel experiments in different cell types using identical methodologies to directly compare NCKAP1's role across contexts.

What are the optimal fixation and antigen retrieval protocols for NCKAP1 immunohistochemistry?

Successful NCKAP1 immunohistochemistry requires careful attention to tissue processing:

Published protocols have successfully demonstrated NCKAP1 expression in paraffin-embedded human brain and liver cancer tissues using a 1:30 dilution . Optimization for each specific tissue type may be necessary, as NCKAP1 expression levels vary significantly across tissues .

How can researchers effectively use NCKAP1 antibodies in co-immunoprecipitation to study protein interactions within the WAVE complex?

NCKAP1 functions within the WAVE complex, making co-immunoprecipitation (co-IP) a crucial technique for understanding its interaction network:

• Lysis buffer optimization: Use buffers that preserve protein-protein interactions:

  • HEPES buffer (pH 7.4) with 150 mM NaCl, 1% NP-40/Triton X-100

  • Include protease and phosphatase inhibitors

  • Mild detergents preserve complex integrity better than harsh detergents

• Cross-linking considerations: Light cross-linking (0.5-1% formaldehyde) can stabilize transient interactions before lysis.

• Antibody selection: Use N-terminal targeting antibodies for co-IP as they have demonstrated efficacy . C-terminal epitopes may be inaccessible within the complex.

• Controls to include:

  • IgG control immunoprecipitation

  • Input sample (pre-IP lysate)

  • Reverse co-IP using antibodies against known partners (WAVE2, Abi-1, CYFIP1)

• Validation approaches:

  • Western blot confirmation of co-precipitated proteins

  • Mass spectrometry for unbiased identification of novel interactors

Since NCKAP1 interacts with multiple proteins in the WAVE complex, researchers should expect co-precipitation of WAVE2, Abi-1, and CYFIP1 in successful experiments .

What considerations are important when using NCKAP1 antibodies in live cell imaging studies?

Live cell imaging studies of NCKAP1 present unique challenges:

• Antibody format: Traditional antibodies cannot penetrate intact cell membranes. Alternative approaches include:

  • Transfection with fluorescently-tagged NCKAP1 constructs

  • Cell-permeable antibody fragments or nanobodies

  • Surface epitope targeting for membrane-exposed regions

• Functional validation: Confirm that tagging or antibody binding doesn't interfere with NCKAP1's function in actin reorganization by performing lamellipodia formation assays.

• Colocalization studies: NCKAP1 should colocalize with known markers:

  • Lamellipodia edge markers (cortactin)

  • WAVE complex components (WAVE2, CYFIP1)

  • Actin cytoskeleton (phalloidin staining)

• Dynamic analysis considerations:

  • NCKAP1 localizes to the cytoplasmic side of lamellipodia membranes

  • Its dynamics should correlate with cell migration and actin remodeling

  • Time-lapse imaging should capture recruitment to leading edges during migration

When studying NCKAP1's role in actin dynamics, researchers should monitor both protein localization and functional outcomes such as lamellipodia formation and cell migration rates.

How does NCKAP1 expression in Alzheimer's disease differ from normal brain tissue, and what methodological approaches best capture these differences?

NCKAP1 expression is markedly reduced in Alzheimer's disease (AD)-affected brains compared to normal brain tissue . Researching this reduction requires specific methodological considerations:

The reduction of NCKAP1 in AD brains suggests a potential role in disease pathogenesis, possibly through disruption of cytoskeletal dynamics essential for neuronal function and synaptic maintenance .

What experimental models best demonstrate the functional consequences of NCKAP1 dysregulation in immune disorders?

While NCKAP1 itself is broadly expressed, its hematopoietic homolog NCKAP1L (HEM-1) plays critical roles in immune system function. Research models for studying these proteins include:

• Human patient samples:

  • Rare NCKAP1L mutations have been identified in patients with immunodeficiency, lymphoproliferation, and hyperinflammation

  • Patient-derived cells show impaired T cell activation and immune synapse morphology

• Mouse models:

  • Nckap1l-deficient mice display anomalies in:

    • Lymphocyte development

    • Phagocytosis

    • Neutrophil migration

• Zebrafish models:

  • Knockdown of nckap1l in zebrafish resulted in defects in neutrophil migration

  • Provides an accessible system for live imaging of immune cell dynamics

• Cell line models:

  • T cell lines for studying activation and immune synapse formation

  • Neutrophil-like cell lines for migration and phagocytosis studies

  • CRISPR/Cas9 engineered knockout lines

Comprehensive investigation should include functional assays measuring:

• T cell activation markers

• Migration assays (Transwell, wound healing)

• Immune synapse formation and morphology

• Actin cytoskeleton reorganization

• Leading edge formation in migrating cells

How can researchers effectively use NCKAP1 antibodies to investigate its potential as a diagnostic biomarker for colorectal cancer metastasis?

Recent research has identified NCKAP1 as a potential biomarker for colorectal cancer metastasis . Effectively investigating this application requires:

In patient-derived xenograft models, NCKAP1 was significantly increased in blood RNA samples, suggesting its potential as a liquid biopsy marker . This non-invasive approach could be particularly valuable for monitoring patients after primary tumor resection.

What emerging technologies could enhance the study of NCKAP1 function and interaction networks?

Several cutting-edge technologies hold promise for advancing NCKAP1 research:

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins can identify transient or weak interactors

  • Allows mapping of NCKAP1's spatial interactome in living cells

  • Can reveal compartment-specific interactions

• Super-resolution microscopy:

  • STORM/PALM techniques provide 10-20 nm resolution

  • Enables visualization of NCKAP1 within lamellipodial complexes

  • Allows precise mapping of dynamic redistribution during cell migration

• CRISPR screening approaches:

  • Genome-wide screens for synthetic lethal interactions

  • Targeted screens of cytoskeletal regulators

  • CRISPRi/CRISPRa for tunable expression modulation

• Single-cell multi-omics:

  • Correlate NCKAP1 expression with transcriptomic/proteomic profiles

  • Identify cell state-specific functions

  • Map expression in heterogeneous tumor environments

• Organoid models:

  • Patient-derived organoids for disease modeling

  • Organ-specific contexts for studying tissue-dependent functions

  • 3D migration and invasion assays

These technologies would help resolve outstanding questions about NCKAP1's context-dependent functions, particularly its apparently contradictory roles in different cancer types .

How can structural biology approaches inform our understanding of NCKAP1 antibody epitope selection?

Structural biology offers valuable insights for NCKAP1 antibody development:

• Structure-guided epitope selection:

  • NCKAP1 forms part of a multi-protein WAVE complex

  • Certain domains may be inaccessible in the assembled complex

  • N-terminal antibodies have proven effective for applications like IP and WB

• Domain-specific targeting:

  • NCKAP1 contains multiple domains with distinct functions

  • Antibodies targeting functional domains can serve as molecular probes

  • Conformation-specific antibodies may distinguish active/inactive states

• Cross-reactivity prediction:

  • Structural comparison with NCKAP1L can identify unique epitopes

  • Sequence alignment across species informs cross-reactivity potential

  • In silico epitope prediction tools guide selection

• Therapeutic development considerations:

  • Function-blocking antibodies could target domains involved in protein-protein interactions

  • Domains involved in EMT regulation represent therapeutic targets

  • Internalization-prone epitopes may enable antibody-drug conjugate approaches

While complete structural information for NCKAP1 remains limited, homology modeling and emerging structural data from related proteins can guide rational antibody development strategies.

What are the methodological challenges in studying NCKAP1's role in both suppressing and promoting tumor growth across different cancer types?

The dual role of NCKAP1 in cancer presents significant research challenges:

• Model system selection:

  • Different cell lines may not recapitulate tissue-specific interaction networks

  • PDX models maintain tumor heterogeneity but are resource-intensive

  • Genetically engineered mouse models can provide tissue context but may not fully replicate human disease

• Expression level considerations:

  • Dose-dependent effects may explain opposing functions

  • Threshold effects versus linear responses require titration experiments

  • Temporal dynamics of expression changes during disease progression

• Pathway cross-talk assessment:

  • In HCC, NCKAP1 affects Rb1/p53 pathways

  • In CRC, NCKAP1 influences EMT markers and migration

  • Comprehensive pathway analysis using phosphoproteomics and transcriptomics

• Technical standardization needs:

  • Consistent antibody validation across studies

  • Standardized functional assays

  • Detailed reporting of cell line genetic backgrounds

• Integrative data analysis:

  • Public database mining for expression correlation with outcomes

  • Multi-cancer type meta-analysis

  • Machine learning approaches for predicting context-dependent functions