NPAS2 Antibody

What is NPAS2 and what cellular functions does it regulate?

NPAS2 is a core circadian molecule that functions as a transcription factor. It plays critical roles in multiple biological processes beyond circadian rhythm regulation. NPAS2 contributes significantly to liver fibrogenesis through direct transcriptional activation of hairy and enhancer of split 1 (Hes1), a critical transcriptor of Notch signaling . In lung adenocarcinoma, NPAS2 binds to and enhances the stability of H2AX mRNA, decreasing tumor cell sensitivity to chemotherapy by augmenting DNA damage repair . NPAS2 also appears to regulate GABAergic neurotransmission in the ventral striatum by binding to genes encoding GABAA receptor subunits . The protein generally forms heterodimers with BMAL1, another core circadian rhythm transcription factor, to regulate the expression of numerous target genes .

Where is NPAS2 primarily expressed in human tissues?

NPAS2 demonstrates variable expression across human tissues. According to the Human Protein Atlas, there is medium consistency between antibody staining and RNA expression data for NPAS2 . In liver fibrosis studies, NPAS2 has been found to be exclusively expressed in activated hepatic stellate cells (aHSCs), with its expression pattern coinciding with that of alpha-smooth muscle actin (α-SMA), a well-established marker of aHSCs in human fibrotic livers . In gastric cancer tissues, immunohistochemical analysis has shown that NPAS2 is mainly expressed in both the cytoplasm and nucleus, with positive cells appearing as yellow and brown granular staining under microscopic examination . NPAS2 mRNA has been detected in various cell lines including MCF-7 (breast cancer) and HCT-15 (colorectal cancer) cells .

How should NPAS2 antibodies be validated before experimental use?

Proper validation of NPAS2 antibodies is essential for reliable research outcomes. Researchers should:

• Confirm antibody specificity using positive and negative control tissues or cells with known NPAS2 expression levels.

• Validate the antibody using multiple detection methods such as Western blot, immunohistochemistry, and immunofluorescence to ensure consistent results.

• Perform siRNA knockdown experiments to confirm antibody specificity, as demonstrated in studies where NPAS2 knockdown was quantitatively determined by qRT-PCR prior to each assay, and only populations with greater than 70% reduction were used in subsequent analyses .

• Cross-validate with mRNA expression data when possible, as seen in liver fibrosis studies where NPAS2 mRNA increase correlated with the induction of α-SMA mRNA (r = 0.458, p = 0.007) .

• Include appropriate controls in each experiment, such as incubation with non-immune IgG as done in ChIP experiments .

How can NPAS2 antibodies be optimized for chromatin immunoprecipitation (ChIP) studies?

ChIP is a powerful technique for studying NPAS2's function as a transcription factor. Based on published research methodologies:

• Select a high-quality ChIP-grade NPAS2 antibody, such as the NPAS2 antibody (H20X, Santa Cruz Biotechnology) used in successful ChIP experiments .

• Include appropriate controls in parallel: anti-acetyl-Histone H3 as a positive control and non-immune rabbit IgG as a negative control .

• For ChIP-Seq applications, follow established protocols but modify fixation time and sonication conditions based on the specific cell type being studied.

• For PCR verification of ChIP results, design primers targeting putative NPAS2 binding sites, such as E-box elements. For example, research has identified an E-box (nucleotide −1875 to nucleotide −1869) in the Hes1 promoter as a critical binding site for NPAS2 .

• Validate ChIP results using site-directed mutagenesis analyses of identified binding sites to confirm their functional significance .

This approach has successfully identified several genes encoding subunits of the GABAA receptor as direct binding targets of NPAS2 and confirmed NPAS2's direct binding to the E-box region of the Hes1 promoter .

What protocol is recommended for NPAS2 immunohistochemistry in cancer tissues?

For optimal immunohistochemical detection of NPAS2 in cancer tissues:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections (typically 30 μm cryostat sections for immediate fixation or 150-300 μm sections for tissue punches) .

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0).

• Primary antibody: Incubate with validated NPAS2 antibody at optimized dilution (determined through titration experiments).

• Detection system: Utilize a highly sensitive detection system compatible with your primary antibody.

• Scoring method: Apply a semi-quantitative integration method for evaluation. Select five high-power visual fields (400×) for each sample under the microscope .

• Interpretation: NPAS2 positive staining appears as yellow and brown granular structures in the cytoplasm and nucleus. Two professional pathologists should independently score the sections without knowledge of clinicopathological factors and clinical outcomes .

Using this approach, researchers have successfully demonstrated significant differences in NPAS2 expression, such as the 65.35% positive rate in gastric cancer tissues compared to 30.69% in adjacent tissues .

How can NPAS2 antibodies be used to study its role in DNA damage response?

To investigate NPAS2's role in DNA damage response:

• Establish experimental cell models:

  • Create NPAS2 knockdown and overexpression systems using siRNA or viral vectors

  • Confirm knockdown efficiency by qRT-PCR and Western blot prior to experiments

• DNA damage induction:

  • Treat cells with DNA-damaging agents such as cisplatin or methyl methanesulfonate (MMS)

  • For MMS treatment, expose cells to 0.015% MMS for 1 hour, wash, and leave under normal conditions for an additional 24 hours

• DNA damage response assessment:

  • Analyze γH2AX accumulation by immunofluorescence and Western blot

  • Perform flow cytometry to determine cell cycle distribution

  • Compare cell phase distribution between normal and NPAS2-depleted cells after DNA damage

• DNA repair capacity evaluation:

  • Use comet assay to measure DNA repair kinetics

  • Assess homology-directed repair (HDR) efficiency using reporter assays

• mRNA stability analysis:

  • Perform RNA immunoprecipitation to examine NPAS2 binding to target mRNAs

  • Use actinomycin D chase experiments to evaluate mRNA half-life changes

Using these approaches, researchers have demonstrated that NPAS2 depletion significantly impairs γH2AX accumulation and homology-directed repair, while NPAS2 can enhance H2AX mRNA stability by direct binding .

How does NPAS2 contribute to liver fibrosis and what experimental approaches can investigate this mechanism?

NPAS2 plays a critical role in liver fibrosis through direct transcriptional activation of Hes1. To investigate this mechanism:

• Animal models: Use both carbon tetrachloride (CCl₄) and bile duct ligation (BDL)-induced fibrosis models with wild-type and NPAS2 knockout mice .

• Fibrosis assessment techniques:

  • Measure hydroxyproline content in liver tissue

  • Evaluate α-SMA and Col1α1 expression by qPCR and Western blot

  • Perform histological analysis to assess liver-bridging fibrosis and collagen deposition

• Cellular mechanistic studies:

  • Isolate primary hepatic stellate cells (HSCs) from wild-type and NPAS2-KO mice

  • Assess HSC activation markers, proliferation, migration, collagen production, and apoptosis

  • Use immortalized human HSC lines (LX2) for complementary in vitro studies

• Molecular mechanism investigation:

  • Perform promoter analysis to identify NPAS2 binding sites in target genes

  • Use site-directed mutagenesis to confirm functional significance of binding sites

  • Conduct ChIP assays to confirm direct binding of NPAS2 to target promoters

These approaches revealed that NPAS2 knockout significantly decreased hydroxyproline content, α-SMA, and Col1α1 expression in fibrotic liver tissues compared to wild-type mice, and NPAS2 directly binds to the E-box region of the Hes1 promoter to activate its transcription .

What are the contradictory findings regarding NPAS2's role in cancer and how can researchers address these contradictions?

The literature presents contradictory findings regarding NPAS2's role in cancer:

To address these contradictions, researchers should:

• Conduct tissue-specific studies:

  • Compare NPAS2 expression and function across different cancer types using the same methodologies

  • Use tissue microarrays to evaluate NPAS2 expression across multiple cancer types simultaneously

• Investigate context-dependent functions:

  • Examine NPAS2's interaction partners in different cellular contexts

  • Study post-translational modifications that might alter NPAS2 function

• Consider temporal aspects:

  • Evaluate NPAS2's role at different stages of cancer progression

  • Account for circadian timing in experimental design

• Perform comprehensive pathway analysis:

  • Use RNA-seq after NPAS2 manipulation to identify tissue-specific downstream targets

  • The more profound effect of NPAS2 knockdown in MCF-7 cells compared to HCT-15 cells suggests its role varies by cancer type

• Use in vivo models:

  • Develop tissue-specific NPAS2 knockout or overexpression models

  • Evaluate tumor growth, metastasis, and response to therapy in these models

How can researchers effectively evaluate the relationship between NPAS2 and circadian rhythm disruption in disease states?

To investigate the relationship between NPAS2, circadian rhythms, and disease:

• Temporal expression analysis:

  • Collect samples at multiple time points across the circadian cycle (e.g., ZT4 and ZT16 as used in previous research)

  • Use qPCR, Western blot, and immunohistochemistry to assess NPAS2 expression patterns

  • Compare expression patterns in normal versus diseased tissues/cells

• Clock gene network analysis:

  • Simultaneously measure multiple clock genes (BMAL1, PER, CRY, ROR, REV-ERB)

  • Analyze the coordinated expression of NPAS2 with other clock components

  • Investigate how NPAS2 functions as both a regulator and target of ROR and REV-ERB

• Functional circadian studies:

  • Use luciferase reporter assays to monitor circadian oscillations

  • Evaluate phase, amplitude, and period changes after NPAS2 manipulation

  • Perform genome-wide circadian transcriptome analysis

• Disease-specific approaches:

  • For liver fibrosis: Compare circadian gene expression in quiescent versus activated HSCs

  • For cancer: Analyze correlations between NPAS2 mutations/polymorphisms and clinical outcomes

  • For neurological disorders: Assess NPAS2-dependent regulation of neurotransmitter receptors

• Translational approaches:

  • Evaluate circadian timing of therapeutic interventions

  • Investigate chronotherapeutic strategies targeting NPAS2 pathways

  • Explore potential of NPAS2 as a biomarker for disease progression or therapeutic response

What techniques can be used to study NPAS2's interaction with BMAL1 and other transcriptional partners?

To investigate NPAS2's protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use NPAS2 antibodies to pull down protein complexes

  • Probe for interaction partners such as BMAL1 using Western blot

  • Perform reciprocal Co-IP using BMAL1 antibodies to confirm interactions

• Proximity ligation assay (PLA):

  • Visualize protein-protein interactions in situ

  • Quantify interactions in different cellular compartments

  • Compare interaction patterns across different time points

• Bimolecular fluorescence complementation (BiFC):

  • Generate fusion constructs of NPAS2 and potential partners with split fluorescent protein fragments

  • Observe reconstituted fluorescence upon protein interaction

  • Track interactions in living cells over time

• ChIP-reChIP:

  • Perform sequential ChIP with NPAS2 and BMAL1 antibodies

  • Identify genomic regions co-occupied by both factors

  • Compare with single ChIP results to determine unique versus shared binding sites

• Mass spectrometry approaches:

  • Use tandem affinity purification followed by mass spectrometry

  • Identify novel interaction partners

  • Characterize post-translational modifications that affect interactions

These approaches can help elucidate how NPAS2 forms heterodimers with BMAL1 to transcriptionally regulate numerous target genes and identify novel interaction partners that may modify NPAS2 function in different cellular contexts.

What are the common technical challenges when using NPAS2 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with NPAS2 antibodies:

• Specificity issues:

  • Validate antibodies using NPAS2 knockout tissues/cells as negative controls

  • Perform pre-absorption tests with recombinant NPAS2 protein

  • Compare results from multiple antibodies targeting different epitopes of NPAS2

• Cross-reactivity with CLOCK protein:

  • Due to structural similarities between NPAS2 and CLOCK, carefully select antibodies with minimal cross-reactivity

  • Confirm specificity by comparing staining patterns in tissues with known differential expression

  • Use siRNA knockdown of NPAS2 versus CLOCK to confirm antibody specificity

• Variable detection sensitivity:

  • Optimize fixation and antigen retrieval protocols for each application

  • Test multiple antibody dilutions and incubation conditions

  • For low expression tissues, consider signal amplification methods

• Temporal expression variations:

  • Account for circadian expression patterns by collecting samples at consistent timepoints

  • When comparing samples, ensure they were collected at the same circadian time

  • Use time-course experiments to capture the full range of expression

• Subcellular localization detection:

  • Use cellular fractionation followed by Western blot to confirm localization patterns

  • For IHC/IF, optimize permeabilization conditions to access nuclear NPAS2

  • Consider the dual nuclear and cytoplasmic localization observed in certain tissues

How can researchers accurately quantify NPAS2 expression levels in tissue samples?

For accurate quantification of NPAS2 expression:

• Immunohistochemistry quantification:

  • Use a semi-quantitative integration method as demonstrated in gastric cancer research

  • Select multiple high-power visual fields (400×) for each sample

  • Have two independent pathologists score the sections

  • Develop consistent scoring criteria based on staining intensity and percentage of positive cells

• Western blot quantification:

  • Use appropriate loading controls (β-actin, GAPDH)

  • Apply densitometric analysis with normalization

  • Include standard curves with recombinant NPAS2 for absolute quantification

  • Consider the potential impact of post-translational modifications on antibody binding

• qRT-PCR for mRNA quantification:

  • Design specific primers that distinguish NPAS2 from related genes

  • Use multiple reference genes for normalization

  • Account for potential alternative splicing by targeting conserved regions

  • Verify correlation between mRNA and protein levels

• Digital pathology approaches:

  • Use automated imaging systems for objective quantification

  • Apply machine learning algorithms to classify positive cells

  • Develop tissue-specific thresholds based on control samples

• Single-cell analysis:

  • Apply single-cell RNA-seq to characterize cell-specific expression patterns

  • Use imaging mass cytometry for simultaneous detection of multiple markers

  • Correlate NPAS2 expression with cell-type specific markers

How might emerging technologies enhance NPAS2 antibody applications in research?

Emerging technologies offer significant potential for advancing NPAS2 research:

• CRISPR-tagged endogenous NPAS2:

  • Generate knock-in cell lines expressing tagged NPAS2 at endogenous levels

  • Avoid artifacts associated with antibody specificity issues

  • Enable live-cell imaging of NPAS2 dynamics

• Single-molecule imaging techniques:

  • Apply super-resolution microscopy to visualize NPAS2 distribution at the nanoscale

  • Use single-molecule tracking to monitor NPAS2 movement and interactions

  • Implement optogenetic approaches to control NPAS2 activity with spatiotemporal precision

• Spatial transcriptomics and proteomics:

  • Map NPAS2 expression and activity across tissue microenvironments

  • Correlate with disease progression in complex tissues

  • Identify cell type-specific roles in heterogeneous samples

• AI-enhanced image analysis:

  • Develop deep learning models for automated quantification of NPAS2 immunostaining

  • Implement multi-parameter analysis to correlate NPAS2 with other markers

  • Create predictive models linking NPAS2 patterns to disease outcomes

• Multiplexed antibody methods:

  • Apply cyclic immunofluorescence or mass cytometry for simultaneous detection of NPAS2 with dozens of other proteins

  • Create comprehensive protein interaction networks

  • Identify context-specific signaling patterns

These technologies could significantly enhance our understanding of NPAS2's role in both normal physiology and disease states, potentially revealing new therapeutic targets and biomarkers.