NDRG1 Antibody, HRP conjugated

What is NDRG1 and why is it an important research target?

NDRG1 (N-myc downstream-regulated gene 1) is a stress-responsive protein involved in hormone responses, cell growth, and differentiation . It plays essential roles in various cellular processes and has been implicated in multiple pathological conditions. NDRG1 is notably important because it functions in cell differentiation, proliferation, and stress responses . The protein is widely expressed across tissue types and undergoes post-translational modifications, particularly phosphorylation, which regulate its activity . NDRG1 has gained significant research interest due to its involvement in cancer progression, where it shows context-dependent functions ranging from tumor suppression to promotion of metastasis depending on the cancer type and stage .

What are the key properties of HRP-conjugated NDRG1 antibodies?

HRP-conjugated NDRG1 antibodies are specialized immunological tools where horseradish peroxidase (HRP) is directly linked to the antibody molecule via the carbohydrate groups of HRP through amine groups . This conjugation eliminates the need for secondary antibodies in detection systems, thereby simplifying experimental procedures. The Phospho-NDRG1 (Thr346) HRP-conjugated antibody typically recognizes NDRG1 when phosphorylated at threonine 346, though it may cross-react with other conserved phosphorylation sites including Thr356 and Thr366 . These antibodies demonstrate specific reactivity across multiple species including human, mouse, rat, and monkey samples with endogenous detection sensitivity . The molecular weight detected is typically around 46-48 kDa, which corresponds to the phosphorylated forms of NDRG1 .

What is the subcellular localization pattern of NDRG1 and how does this affect antibody selection?

NDRG1 exhibits complex subcellular localization across the cell membrane, nucleus, and cytoplasm . The subcellular distribution pattern can vary depending on cellular context, disease state, and phosphorylation status. Recent research suggests that NDRG1 subcellular localization may have prognostic value in certain cancers . When selecting an NDRG1 antibody, researchers should consider both the specific subcellular compartment of interest and the phosphorylation status they wish to detect. For instance, phosphorylated NDRG1 may show different localization patterns compared to the unphosphorylated form. For comprehensive analyses, researchers might need to employ subcellular fractionation techniques alongside immunoblotting to accurately track NDRG1 localization across cellular compartments . The HRP-conjugated phospho-specific antibodies are particularly useful when studying the functional significance of NDRG1 phosphorylation in different subcellular locations.

What are the optimal conditions for using HRP-conjugated NDRG1 antibodies in Western blotting?

For optimal Western blotting results with HRP-conjugated NDRG1 antibodies, several critical parameters must be carefully controlled:

For phosphorylated NDRG1 detection, samples treated with agents that activate SGK1 (which phosphorylates NDRG1 at multiple sites including Thr346) can serve as positive controls .

How can researchers optimize immunofluorescence protocols using NDRG1 antibodies?

While HRP-conjugated antibodies are not typically used for immunofluorescence, researchers studying NDRG1 localization often need to optimize immunofluorescence protocols. For studying NDRG1 in cellular contexts:

• Fixation method: 4% paraformaldehyde for 15 minutes followed by permeabilization with 0.1% Triton X-100 works effectively for NDRG1 detection.

• Antibody selection: For co-localization studies, select unconjugated primary antibodies against NDRG1 and potential interaction partners like GSK-3β or Nur77 . Research has demonstrated that NDRG1 interacts with these proteins, affecting pathways like β-catenin signaling.

• Controls: Include controls for antibody specificity by using NDRG1 knockdown cells.

• Image analysis: Quantitative co-localization analysis should be performed using appropriate software (e.g., ImageJ with co-localization plugins).

• Validation across conditions: Confirm localization patterns under different cellular stresses, as NDRG1 is stress-responsive and its localization may change under hypoxia or other stress conditions .

Research has successfully used immunofluorescence to demonstrate that NDRG1 suppression dramatically decreases β-catenin levels and prevents its nuclear accumulation, highlighting the utility of this technique in studying NDRG1's functional interactions .

What methodological approaches can be used to study NDRG1 phosphorylation and its functional significance?

NDRG1 phosphorylation, particularly at sites like Thr346, Thr356, and Thr366, plays crucial roles in regulating its function. To comprehensively study this:

• Phospho-specific antibody selection: Use antibodies like Phospho-NDRG1 (Thr346) that specifically recognize phosphorylated forms .

• Kinase manipulation: Modulate the activity of upstream kinases like SGK1 or GSK-3β through inhibitors, activators, or genetic approaches to assess effects on NDRG1 phosphorylation .

• Phosphorylation site mutants: Generate and express NDRG1 constructs with mutations at key phosphorylation sites (e.g., T346A) to determine the functional significance of specific phosphorylation events.

• Mass spectrometry approaches: Employ phosphoproteomic analysis to identify all phosphorylation sites and their relative abundance under different conditions. The Phospho-NDRG1 (Thr346) site was originally discovered using PhosphoScan®, an LC-MS/MS platform for modification site discovery .

• Functional correlation: Correlate phosphorylation status with functional outcomes such as protein-protein interactions, subcellular localization, and downstream signaling events.

• In vivo models: Utilize animal models with targeted NDRG1 mutations to assess the physiological relevance of phosphorylation events.

Research has shown that NDRG1 is phosphorylated by SGK1 at multiple sites (Thr328, Ser330, Thr346, Thr356, and Thr366), and this phosphorylation primes NDRG1 for subsequent phosphorylation by GSK-3 , highlighting the complex regulatory mechanisms controlling NDRG1 function.

How can NDRG1 antibodies be applied to investigate its role in cancer progression mechanisms?

NDRG1 has emerged as a key player in cancer biology with context-dependent roles. To effectively investigate these functions:

The table below summarizes key findings on NDRG1's role in cancer progression:

What is the significance of NDRG1 in vascular biology and how can researchers investigate this aspect?

NDRG1 plays critical roles in vascular biology, particularly in endothelial inflammation and remodeling . To effectively study these functions:

• Endothelial cell models: Use HRP-conjugated NDRG1 antibodies to study expression in various endothelial cell types under normal and pathological conditions (e.g., inflammatory cytokine stimulation).

• Loss-of-function studies: Employ NDRG1 knockdown approaches (e.g., shRNA, CRISPR-Cas9) to assess functional consequences in endothelial cells .

• Inflammatory marker correlation: Analyze how NDRG1 expression correlates with inflammatory markers. Research has demonstrated that NDRG1 knockdown substantially attenuates IL-1β and TNF-α-induced expression of cytokines/chemokines and adhesion molecules .

• Thrombosis assays: Investigate NDRG1's role in coagulation pathways, as studies show NDRG1 inhibition attenuates expression of procoagulant molecules (PAI-1, TF) while increasing antithrombotic factors (TM, t-PA) .

• Mechanistic pathway analysis: Explore NDRG1's interaction with Nur77 and effects on NF-κB, MAPK signaling, and AP-1 transcriptional activity in vascular contexts .

• In vivo models: Utilize endothelial cell-specific NDRG1 knockout mice to study effects on neointima formation, atherosclerosis, and arterial thrombosis .

Research has demonstrated that NDRG1 expression is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions, highlighting its relevance to vascular pathology .

How can researchers resolve contradictory findings regarding NDRG1's role as tumor suppressor versus oncogene?

The literature presents conflicting views on NDRG1's role in cancer, functioning as either a tumor suppressor or oncogene depending on context. To address these contradictions:

• Context-specific analysis: Systematically analyze NDRG1 expression and function across different cancer types, stages, and genetic backgrounds using phospho-specific and total NDRG1 antibodies.

• Cellular stress conditions: Examine NDRG1's role under different stress conditions (hypoxia, nutrient deprivation, etc.), as its function may change depending on the cellular environment .

• Interactome analysis: Use co-immunoprecipitation with NDRG1 antibodies followed by mass spectrometry to identify context-specific interaction partners that might explain divergent functions.

• Subcellular localization studies: Investigate whether differential subcellular localization correlates with opposing functions using fractionation and immunofluorescence approaches .

• Post-translational modification profiling: Examine how different patterns of phosphorylation or other modifications affect NDRG1 function using phospho-specific antibodies and mutagenesis approaches.

• Isoform-specific analysis: Determine whether different NDRG1 isoforms (up to 3 have been reported ) have distinct functions in cancer progression.

Research in hepatocellular carcinoma showed that NDRG1 promotes tumor growth by binding to GSK-3β and Nur77, preventing β-catenin degradation , while other studies in different contexts have suggested tumor-suppressive functions. These contradictions highlight the importance of comprehensive, context-specific analysis.

What are common troubleshooting strategies for inconsistent NDRG1 detection in Western blotting?

Researchers frequently encounter challenges when detecting NDRG1, particularly its phosphorylated forms. Here are key troubleshooting approaches:

• Multiple bands detection: NDRG1 can appear as multiple bands around 46-48 kDa due to post-translational modifications . To distinguish specific signals:

  • Compare with positive controls and NDRG1 knockdown samples

  • Use phosphatase treatment of some samples to identify phosphorylation-dependent bands

  • Run longer SDS-PAGE gels for better separation of closely migrating forms

• Weak signal detection:

  • Increase protein loading (30-50 μg total protein recommended)

  • Reduce antibody dilution (1:500 instead of 1:1000)

  • Extend exposure time during detection

  • Use enhanced chemiluminescence substrates for higher sensitivity

  • Ensure samples contain phosphatase inhibitors for phospho-specific detection

• High background issues:

  • Increase blocking time or concentration (5% BSA in TBST for 2 hours)

  • More extensive washing steps (5-6 washes of 10 minutes each)

  • Dilute antibody in fresh blocking solution

  • Filter blocking and antibody solutions to remove particulates

• Sample preparation considerations:

  • For phosphorylated NDRG1 detection, flash-freeze tissues immediately after collection

  • Include both phosphatase inhibitors and protease inhibitors in lysis buffers

  • Process samples consistently at cold temperatures to prevent degradation

• Signal normalization strategies:

  • Always include loading controls (β-actin, GAPDH)

  • For phospho-specific detection, normalize to total NDRG1 levels in addition to loading controls

How should researchers interpret differences between phosphorylated and total NDRG1 signal patterns?

Interpreting the relationship between phosphorylated and total NDRG1 signals requires careful consideration:

• Relative changes interpretation:

  • Increased phospho-NDRG1 without total NDRG1 changes indicates activation of upstream kinases (e.g., SGK1)

  • Parallel increases in both phospho and total NDRG1 suggest transcriptional/translational upregulation plus pathway activation

  • Decreased phospho-NDRG1 with stable total levels indicates pathway inhibition or phosphatase activation

• Multiple phosphorylation site analysis:

  • NDRG1 is phosphorylated at multiple sites (Thr328, Ser330, Thr346, Thr356, Thr366)

  • Differential phosphorylation patterns may indicate distinct signaling events

  • Consider examining multiple phosphorylation sites when possible

• Functional correlation approaches:

  • Correlate phosphorylation status with interaction partners (e.g., GSK-3β, Nur77)

  • Assess downstream effects such as β-catenin levels and nuclear localization

  • Evaluate cellular phenotypes (proliferation, migration) in relation to phosphorylation status

• Temporal dynamics considerations:

  • Phosphorylation events often precede functional changes

  • Consider time-course experiments to capture the sequence of events

  • Rapid phosphorylation changes may be missed in steady-state analyses

• Microenvironmental influence assessment:

  • Hypoxia can induce NDRG1 expression and alter its phosphorylation status

  • Consider analyzing samples from different microenvironmental conditions within tumors

Research has shown that NDRG1 phosphorylation by SGK1 primes it for subsequent phosphorylation by GSK-3 , illustrating the complex interplay between different phosphorylation events that must be considered during data interpretation.

How can researchers validate the specificity of NDRG1 antibody signals across different experimental systems?

Ensuring antibody specificity is crucial for reliable NDRG1 research. Comprehensive validation should include:

• Genetic knockout/knockdown controls:

  • Use NDRG1 knockdown samples (siRNA, shRNA) as negative controls

  • CRISPR-Cas9 generated knockout cell lines provide definitive negative controls

  • Rescue experiments with exogenous NDRG1 expression can confirm specificity

• Cross-reactivity assessment:

  • Test antibody against related NDRG family members (NDRG2, NDRG3, NDRG4)

  • Compare reactivity patterns across species (human, mouse, rat) known to have different levels of conservation

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application to samples

  • Signal should be blocked or significantly reduced if antibody is specific

• Correlation between detection methods:

  • Compare results between different antibodies targeting different NDRG1 epitopes

  • Correlate protein detection with mRNA levels (qPCR, RNA-seq)

  • Use orthogonal techniques (mass spectrometry) for protein identification

• Phospho-specificity validation:

  • Treat samples with phosphatases to confirm phospho-specific antibody reactivity

  • Use phosphomimetic and phospho-null NDRG1 mutants (e.g., T346E and T346A)

  • Compare reactivity in samples with activated versus inhibited upstream kinases

• Cross-platform validation:

  • Confirm findings across multiple techniques (Western blot, immunofluorescence, IHC)

  • Evaluate antibody performance in different sample types (cell lines, tissues, species)

A thorough validation approach ensures that observed signals truly represent NDRG1 and its modifications, preventing misinterpretation of experimental results.

How can researchers utilize NDRG1 antibodies to explore its role in cancer stem cell biology?

Recent research has revealed NDRG1's critical role in cancer stem cell (CSC) regulation, particularly in hepatocellular carcinoma . To effectively investigate this emerging area:

• CSC marker co-expression analysis:

  • Use multicolor immunofluorescence to analyze co-expression of NDRG1 with established CSC markers like EpCAM

  • Apply flow cytometry with NDRG1 antibodies to isolate and characterize CSC populations

  • Perform single-cell analysis to identify NDRG1-high subpopulations within heterogeneous tumors

• Functional CSC assays:

  • Analyze sphere formation capacity in NDRG1-manipulated cells (overexpression, knockdown)

  • Conduct limiting dilution assays in vivo to assess tumor-initiating capacity

  • Evaluate chemoresistance profiles in relation to NDRG1 expression

• Protein stabilization mechanisms:

  • Investigate NDRG1's role in stabilizing CSC markers like EpCAM through:

    • Co-immunoprecipitation to confirm protein-protein interactions

    • Ubiquitination assays to assess NDRG1's impact on protein degradation

    • Half-life studies using cycloheximide chase experiments

• Lineage tracing approaches:

  • Develop NDRG1 reporter systems to track CSC populations in real-time

  • Use inducible NDRG1 knockout/knockin models to assess CSC dynamics

Research has demonstrated that NDRG1 enhances CSC expansion through regulation of EpCAM stability, and NDRG1 knockdown inhibits self-renewal of tumor-initiating cells . This highlights NDRG1 as a promising target for therapies aimed at eliminating cancer stem cells.

What methodological considerations should be made when exploring NDRG1's role in vascular inflammation and thrombosis?

NDRG1's involvement in vascular inflammation and thrombosis represents an important research direction . Key methodological approaches include:

• Endothelial cell model selection:

  • Use primary human endothelial cells from different vascular beds (HUVEC, HAEC, HMVEC)

  • Consider immortalized cell lines for mechanistic studies

  • Employ 3D vascular models or microfluidic systems for physiologically relevant contexts

• Inflammatory induction protocols:

  • Compare NDRG1 responses to different inflammatory stimuli (IL-1β, TNF-α, LPS)

  • Conduct time-course studies to capture dynamic changes in NDRG1 expression and phosphorylation

  • Combine with shear stress models to mimic physiological vascular conditions

• Transcription factor interaction analysis:

  • Investigate NDRG1's interactions with Nur77 and NF-κB using:

    • Chromatin immunoprecipitation (ChIP) assays

    • Reporter gene assays for transcriptional activity

    • Co-immunoprecipitation for protein-protein interactions

• Thrombosis-related assays:

  • Measure expression of procoagulant (PAI-1, TF) and anticoagulant (TM, t-PA) factors

  • Conduct functional coagulation assays in NDRG1-manipulated cells

  • Use in vivo thrombosis models with endothelial-specific NDRG1 knockout mice

• Signaling pathway dissection:

  • Analyze MAPK activation, c-Jun phosphorylation, and AP-1 activity

  • Use specific pathway inhibitors to delineate the hierarchy of signaling events

  • Employ phospho-specific antibodies to track activation states of key intermediates

Research has shown that endothelial cell-specific NDRG1 knockout mice exhibit markedly attenuated neointima and atherosclerosis formation, as well as reduced arterial thrombosis compared to wild-type littermates , highlighting the translational potential of targeting NDRG1 in vascular diseases.

How can phospho-specific NDRG1 antibodies contribute to understanding its differential functions across tissue types?

NDRG1 exhibits tissue-specific functions that may be regulated through differential phosphorylation patterns. To explore this complexity:

• Comparative tissue profiling:

  • Create phosphorylation maps of NDRG1 across healthy and diseased tissues using phospho-specific antibodies

  • Compare total NDRG1 versus site-specific phosphorylation patterns

  • Correlate with expression of known kinases (SGK1, GSK-3β) across tissues

• Kinase activity manipulation:

  • Use tissue-specific conditional knockout models for relevant kinases

  • Apply selective kinase inhibitors in different cellular models

  • Analyze resulting changes in NDRG1 phosphorylation and function

• Phosphorylation-dependent interactome analysis:

  • Perform phospho-specific pulldowns followed by mass spectrometry

  • Compare interaction partners of differently phosphorylated NDRG1 forms

  • Validate key interactions using co-immunoprecipitation and functional assays

• Multi-omics integration approaches:

  • Correlate phospho-NDRG1 patterns with transcriptomic and proteomic data

  • Identify tissue-specific signaling networks centered around NDRG1

  • Develop computational models predicting context-dependent functions

• In vivo phosphorylation site mutant models:

  • Generate knock-in models with phospho-null or phospho-mimetic mutations

  • Analyze phenotypic consequences in different tissues

  • Compare with pharmacological manipulation of relevant kinases

Understanding the phosphorylation-dependent functions of NDRG1 across tissues may help reconcile apparently contradictory roles reported in different contexts and could inform tissue-specific therapeutic approaches targeting NDRG1-related pathways.