NEIL1 Antibody

What is NEIL1 and what are its primary cellular functions?

NEIL1 (nei like DNA glycosylase 1) is a DNA repair enzyme belonging to the FPG protein family with a molecular weight of 43.7 kDa and 390 amino acid residues in humans. It primarily functions in base excision repair (BER) of DNA damaged by oxidation or mutagenic agents. NEIL1 is localized in both the nucleus and cytoplasm and is ubiquitously expressed across multiple tissue types. Its primary role involves initiating prereplicative repair of oxidized DNA bases in the replicating genome, thereby preventing mutagenic replication .

How is NEIL1 involved in DNA repair mechanisms?

NEIL1 initiates prereplicative base excision repair by recognizing and removing oxidized bases in DNA. It forms multiprotein "BERosome" complexes with DNA replication proteins via its C-terminal domain (CTD), allowing recruitment at the replication fork. These interactions are crucial for efficient BER in mammalian cells. NEIL1 physically interacts with replication proteins including proliferating cell nuclear antigen clamp loader replication factor C (RFC), DNA polymerase δ (Polδ), and DNA ligase I (LigI). Notably, RFC interaction results in approximately 8-fold stimulation of NEIL1's DNA glycosylase activity .

What structural domains of NEIL1 are critical for its function?

The C-terminal domain (CTD) of NEIL1 is essential for its interactions with replication and repair proteins. This disordered region mediates binary interactions with various proteins in the BERosome complex. The catalytic domain is critical for suppression of IFN-β production in viral infection contexts, although interestingly, the enzymatic activity itself is dispensable for this function. Disruption of the CTD, as observed in deletion mutants (e.g., N311), inhibits complete BER in vitro and prevents chromatin association and recruitment at replication foci in S phase cells .

What criteria should be considered when selecting a NEIL1 antibody for research?

When selecting a NEIL1 antibody, researchers should consider: (1) Antibody type (monoclonal vs. polyclonal) - monoclonals offer higher specificity while polyclonals provide stronger signals; (2) Host species - important for avoiding cross-reactivity in multi-label experiments; (3) Target species reactivity - ensure the antibody recognizes NEIL1 in your experimental organism (human, mouse, rat, etc.); (4) Application compatibility - verify validation for your intended application (WB, IF, IHC, ELISA, etc.); (5) Target epitope - antibodies targeting different regions may yield different results; (6) Published validation data - prefer antibodies with peer-reviewed citations demonstrating specificity and reproducibility .

How can I validate a NEIL1 antibody for specificity in my experimental system?

A comprehensive NEIL1 antibody validation should include: (1) Positive controls using cell lines known to express NEIL1 (e.g., HeLa cells); (2) Negative controls via NEIL1 knockdown/knockout cells; (3) Peptide competition assay to confirm epitope specificity; (4) Western blot analysis to verify single band at expected molecular weight (43.7 kDa); (5) Immunofluorescence co-localization with established nuclear/DNA repair markers; (6) Cross-validation with multiple antibodies targeting different NEIL1 epitopes; and (7) Comparison of detection patterns with published NEIL1 expression profiles across tissues. This multi-approach validation ensures reliable antibody performance in subsequent experiments .

What are the common pitfalls in NEIL1 antibody-based experiments?

Common pitfalls include: (1) Inadequate antibody validation leading to non-specific signals; (2) Incorrect antibody dilution resulting in background issues or weak signals; (3) Cell fixation methods affecting epitope accessibility, particularly for nuclear NEIL1 detection; (4) Cross-reactivity with other FPG family members due to sequence homology; (5) Insufficient blocking leading to non-specific binding; (6) Assuming subcellular localization without proper controls (NEIL1 distributes between nucleus and cytoplasm); (7) Misinterpreting altered NEIL1 levels in stress conditions without appropriate normalization; and (8) Overlooking potential post-translational modifications that might affect antibody recognition .

What are the optimal conditions for detecting NEIL1 using Western blotting?

For optimal NEIL1 detection via Western blotting: (1) Sample preparation: Use RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors, and N-ethylmaleimide to preserve post-translational modifications; (2) Protein loading: Load 20-50 μg of nuclear extract or 50-75 μg of whole cell lysate; (3) Separation: Use 10-12% SDS-PAGE gels for optimal resolution of the 43.7 kDa NEIL1 protein; (4) Transfer: Semi-dry transfer at 15V for 45 minutes or wet transfer at 30V overnight at 4°C; (5) Blocking: 5% non-fat milk in TBST for 1 hour at room temperature; (6) Primary antibody: Incubate at 1:500-1:1000 dilution overnight at 4°C; (7) Detection: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence. Expect bands at approximately 43.7 kDa, with potential additional bands representing post-translationally modified forms .

How can I effectively use NEIL1 antibodies for immunofluorescence studies?

For successful NEIL1 immunofluorescence: (1) Cell preparation: Culture cells on poly-L-lysine coated coverslips at 60-70% confluence; (2) Fixation: 4% paraformaldehyde for 15 minutes followed by permeabilization with 0.2% Triton X-100 for 10 minutes (avoid methanol fixation as it may disrupt nuclear architecture); (3) Blocking: 3% BSA in PBS for 1 hour; (4) Primary antibody: Incubate NEIL1 antibody at 1:100-1:200 dilution overnight at 4°C; (5) Secondary antibody: Fluorophore-conjugated secondary at 1:500 for 1 hour at room temperature; (6) Counterstaining: DAPI for nuclear visualization; (7) Controls: Include NEIL1 knockdown controls and co-staining with markers of replication (PCNA) or DNA damage (γH2AX) to validate localization; (8) Imaging: Confocal microscopy with z-stack acquisition for accurate nuclear localization assessment .

What methods can be used to study NEIL1 protein interactions in cells?

To investigate NEIL1 protein interactions: (1) Co-immunoprecipitation: Use anti-NEIL1 antibodies to pull down NEIL1 and associated proteins, followed by Western blotting for suspected interaction partners (RFC, Polδ, LigI); (2) Proximity ligation assay (PLA): Visualize and quantify NEIL1 interactions with replication/repair proteins with subcellular resolution; (3) FRET/BRET analysis: For studying dynamic interactions in living cells; (4) GST pull-down assays: With recombinant NEIL1 domains to map interaction interfaces; (5) Chromatin immunoprecipitation (ChIP): To identify NEIL1 association with specific DNA regions; (6) Size exclusion chromatography: To isolate NEIL1-containing multiprotein complexes from cell extracts; (7) Mass spectrometry: For unbiased identification of novel NEIL1 interaction partners; (8) Yeast two-hybrid screening: To identify additional interaction partners. These methods should be used in combination for comprehensive interaction mapping .

How does NEIL1 coordinate with other BER proteins during DNA repair?

NEIL1 coordinates with other BER proteins through a complex network of interactions mediated primarily through its C-terminal domain (CTD). After recognizing and excising oxidized bases, NEIL1 recruits AP endonuclease 1 (APE1) for processing the resulting abasic site. NEIL1 directly interacts with DNA polymerase β (Polβ) for gap filling, and with DNA ligase IIIα/XRCC1 or DNA ligase I for nick sealing. In replicating cells, NEIL1 forms a "BERosome" complex with replication factors including RFC, Polδ, and LigI. This complex coordination ensures efficient repair synthesis during replication. Disruption of these interactions, as seen with CTD deletion mutants, significantly impairs complete BER, highlighting the importance of these protein-protein interactions in the BER pathway .

What is the significance of NEIL1's prereplicative repair activity?

NEIL1's prereplicative repair activity is critical for genomic stability during DNA replication. NEIL1 acts as a "cow-catcher" ahead of the replication fork, recognizing and removing oxidized bases in the template strand before the replication machinery encounters them. This activity prevents replication blocks, mutagenic nucleotide incorporation, and potential double-strand break formation that could occur if the replication fork encountered unrepaired DNA damage. Experimental evidence shows that NEIL1 associates with the replication apparatus through interactions with RFC, Polδ, and LigI. This strategic positioning allows for efficient scanning and repair of oxidative DNA damage in the replicating genome, maintaining replication fidelity and preventing the perpetuation of mutations in daughter cells .

How do post-translational modifications regulate NEIL1 activity?

Post-translational modifications (PTMs) of NEIL1 dynamically regulate its enzymatic activity, substrate specificity, protein interactions, and subcellular localization throughout the cell cycle and in response to oxidative stress. Key PTMs include: (1) Phosphorylation: Cell cycle-dependent phosphorylation by CDKs and checkpoint kinases modulates NEIL1's activity, with hyperphosphorylation during S-phase enhancing its prereplicative repair function; (2) Acetylation: Affects NEIL1's DNA binding affinity and catalytic activity; (3) Ubiquitination: Regulates NEIL1 protein levels and potentially its localization; (4) SUMOylation: May influence NEIL1's interaction with other repair proteins. These modifications create a complex regulatory network that fine-tunes NEIL1 activity according to cellular requirements. Importantly, aberrant modifications may contribute to pathological conditions by disrupting normal NEIL1 function in DNA damage repair .

What role does NEIL1 play in influenza virus infection?

NEIL1 plays a dual role in influenza A virus (IAV) infection: enhancing viral replication while suppressing host immune response. As identified through high-throughput sgRNA screening, NEIL1 acts as a critical regulator of interferon-beta (IFN-β) production during viral infection. It promotes IAV replication by regulating methylation of the cytonuclear IFN-β promoter (particularly at CpG-345), thereby inhibiting expression of IFN-β and IFN-stimulating genes. Additionally, NEIL1 directly enhances viral replication by interacting with the N- and C-terminus of IAV nucleoprotein (NP), facilitating NP entry into the nucleus and enhancing stability of the viral ribonucleoprotein (vRNP) complex. These functions collectively contribute to IAV replication, transcription, and immune evasion .

How does NEIL1 regulate IFN-β production during viral infection?

NEIL1 negatively regulates IFN-β production during viral infection through an enzyme-independent mechanism primarily involving methylation regulation. It specifically targets the cytonuclear IFN-β promoter, with CpG-345 being a key methylation site. The catalytic domain of NEIL1 is essential for this suppression, although interestingly, its enzymatic activity is dispensable. This suggests NEIL1 likely functions through protein-protein interactions rather than direct DNA glycosylase activity. By modulating methylation patterns at the IFN-β promoter, NEIL1 creates an epigenetic environment that inhibits transcription of this crucial antiviral cytokine. This suppression subsequently reduces expression of IFN-stimulated genes, compromising the host's antiviral response and creating a more favorable environment for viral replication and spread .

How can CRISPR-Cas9 be used to study NEIL1 function?

CRISPR-Cas9 offers powerful approaches for investigating NEIL1 function through: (1) Complete knockout: Generate NEIL1-null cell lines to study phenotypic consequences on DNA repair capacity and viral susceptibility; (2) Domain-specific mutations: Create precise modifications to functional domains (catalytic domain, CTD) to dissect their specific roles; (3) Endogenous tagging: Insert fluorescent protein tags or epitope tags to track native NEIL1 localization and dynamics; (4) Promoter modification: Alter NEIL1 expression levels or introduce inducible elements; (5) Single nucleotide variants: Recreate disease-associated NEIL1 variants to study functional impacts; (6) High-throughput screening: Perform genome-wide screens in NEIL1-deficient backgrounds to identify synthetic lethal interactions or compensatory pathways. Experimental design should include validation of editing efficiency via sequencing, protein expression analysis, off-target assessment, and careful selection of appropriate control cell lines .

What are the best approaches for studying NEIL1's role in oxidative DNA damage repair?

To comprehensively investigate NEIL1's role in oxidative DNA damage repair: (1) DNA damage induction: Use site-specific oxidative damage induction via photosensitizers (Ro19-8022) or microirradiation techniques; (2) Repair kinetics assessment: Employ alkaline comet assay, pulse-field gel electrophoresis, or immunostaining for 8-oxoG to quantify damage and repair rates; (3) Substrate specificity analysis: Utilize synthetic oligonucleotides containing specific oxidative lesions (8-oxoG, FapyG, thymine glycol) in enzymatic assays; (4) Real-time repair visualization: Apply FRAP (Fluorescence Recovery After Photobleaching) with fluorescently-tagged NEIL1 to analyze recruitment kinetics; (5) Replication-coupled repair: Use DNA fiber analysis and iPOND (isolation of Proteins On Nascent DNA) to study NEIL1's prereplicative function; (6) Structure-function analysis: Perform in vitro reconstitution assays with purified NEIL1 variants and BER components .

How can differential expression of NEIL1 across tissue types be accurately quantified?

For accurate quantification of NEIL1 expression across tissues: (1) RNA analysis: Employ RT-qPCR with carefully designed primers spanning exon junctions to detect specific NEIL1 transcript variants; RNA-seq for broader transcriptomic context; (2) Protein analysis: Use validated NEIL1 antibodies for Western blotting with tissue-specific positive and negative controls; (3) Tissue microarrays: Perform immunohistochemistry on multiple tissue samples simultaneously with standardized protocols; (4) Single-cell approaches: Apply scRNA-seq or CyTOF to identify cell type-specific expression patterns within heterogeneous tissues; (5) Normalization strategy: Utilize multiple reference genes or proteins validated for stability across the specific tissues being compared; (6) Spatial analysis: Implement in situ hybridization or spatial transcriptomics to map NEIL1 expression within tissue architecture; (7) Cross-validation: Compare protein and mRNA levels to identify potential post-transcriptional regulation mechanisms across tissues .

What is the relationship between NEIL1 polymorphisms and disease susceptibility?

NEIL1 genetic variants have been associated with several disease conditions through mechanisms involving altered DNA repair capacity. The G83D polymorphism affects glycosylase activity and has been linked to increased risk of gastric and colorectal cancers. The rs5745908 variant correlates with metabolic syndrome risk, while rs11634109 is associated with primary open-angle glaucoma. Mechanistically, these polymorphisms may alter NEIL1's catalytic efficiency, substrate specificity, protein interactions, or stability. Disease association studies require careful experimental design, including genotyping validation, population stratification control, and functional characterization of variant proteins. Researchers should employ both case-control studies and functional assays to establish causative relationships between NEIL1 variants and disease susceptibility .

How might NEIL1-targeted approaches be developed for cancer therapy?

NEIL1-targeted approaches for cancer therapy could exploit several strategies: (1) Synthetic lethality: Develop inhibitors targeting NEIL1 in cancers with defects in complementary DNA repair pathways; (2) Radiosensitization: Use NEIL1 inhibitors as adjuvants to enhance radiation therapy effectiveness by preventing repair of oxidative DNA damage; (3) CTD peptide mimetics: Employ dominant-negative inhibition using NEIL1 C-terminal domain peptides to disrupt the BERosome, selectively targeting rapidly dividing cancer cells; (4) Combination therapy: Pair NEIL1 inhibitors with chemotherapeutics that induce oxidative stress; (5) Cancer-specific delivery: Develop tumor-targeted delivery systems for NEIL1 inhibitors to minimize effects on normal tissues. Preclinical development should include comprehensive toxicity assessment, pharmacokinetic/pharmacodynamic modeling, and identification of appropriate biomarkers for patient stratification .

What are the implications of NEIL1 dysfunction in neurodegenerative disorders?

NEIL1 dysfunction may contribute to neurodegenerative disorders through accumulation of oxidative DNA damage in non-dividing neuronal cells. The brain is particularly vulnerable to oxidative stress due to high oxygen consumption, enrichment of peroxidizable lipids, and limited antioxidant capacity. NEIL1 is crucial for repairing oxidized bases like 8-oxoguanine and FapyG that accumulate in aging and neurodegenerative conditions. Research methods to investigate this connection include: (1) Analysis of NEIL1 expression/activity in post-mortem brain tissues from patients with Alzheimer's, Parkinson's, or ALS; (2) Measurement of oxidative DNA damage biomarkers in NEIL1-deficient model systems; (3) Age-dependent behavioral studies in NEIL1 knockout mice; (4) Single-cell transcriptomics to identify neural cell populations most affected by NEIL1 deficiency; (5) Development of neuron-specific NEIL1 modulation to assess causality in disease models .

How should researchers design experiments to study NEIL1 activity in different cellular contexts?

A comprehensive experimental design for studying NEIL1 activity across cellular contexts should include: (1) Cell type selection: Compare primary cells, immortalized lines, and disease-relevant models; (2) Physiological conditions: Examine NEIL1 under normal conditions, oxidative stress, viral infection, and replication stress; (3) Activity assays: Measure enzymatic activity using fluorescence-based glycosylase assays and AP lyase assays; (4) Cellular compartmentalization: Assess nuclear vs. cytoplasmic NEIL1 activity using subcellular fractionation; (5) Protein complex analysis: Compare NEIL1 activity in isolation vs. in multiprotein complexes; (6) Cell cycle synchronization: Evaluate phase-specific activity differences; (7) Controls: Include NEIL1 catalytic mutants (P2T, E3Q) and CTD deletion mutants; (8) Data analysis: Apply appropriate statistical tests with multiple biological replicates (n≥3) and consider potential confounding variables including cell confluence and passage number .

What statistical approaches are most appropriate for analyzing NEIL1 antibody-based experimental data?

For NEIL1 antibody-based experimental data analysis: (1) Western blot quantification: Use normalized band intensity with appropriate loading controls, applying ANOVA with post-hoc tests for multi-group comparisons or t-tests for paired comparisons; (2) Immunofluorescence quantification: Employ automated image analysis software with rigorous thresholding and cell segmentation, analyzed via non-parametric tests if intensity distributions are non-normal; (3) Co-localization analysis: Calculate Pearson's or Mander's coefficients with appropriate controls; (4) Reproducibility: Perform experiments with ≥3 biological replicates, reporting means with standard error; (5) Sample size determination: Conduct power analysis based on expected effect sizes; (6) Outlier handling: Apply consistent criteria for exclusion; (7) Multiple comparison correction: Use Bonferroni or FDR methods when testing multiple hypotheses; (8) Data visualization: Present paired data points alongside means, utilize box plots showing distribution characteristics .

How can researchers reconcile contradictory findings regarding NEIL1 function in different experimental systems?

To reconcile contradictory findings about NEIL1 function: (1) Methodological analysis: Carefully compare experimental protocols, antibody specifications, cell lines, and assay conditions between studies; (2) Cell type considerations: Recognize that NEIL1 function may differ between cancer cell lines, primary cells, and in vivo systems; (3) Context dependency: Evaluate whether differences relate to specific cellular conditions (oxidative stress levels, replication status, etc.); (4) Protein interaction variations: Assess whether contradictions stem from differential protein partner availability across systems; (5) Isoform expression: Confirm which NEIL1 splice variants predominate in each system; (6) Post-translational modifications: Determine whether differences in PTM profiles could explain functional variations; (7) Direct replication attempts: Perform side-by-side experiments under identical conditions; (8) Meta-analysis approach: Systematically compare multiple studies to identify patterns explaining apparent contradictions; (9) Collaborative resolution: Establish collaborations between labs reporting different findings to exchange materials and protocols .

Table 1: Comparison of NEIL1 Antibody Applications in Research

Table 2: NEIL1 Domain Structure and Function