NBL1 Antibody

What is NBL1 and what are its known biological functions?

NBL1, also known as DAN, DAND1, or Neuroblastoma suppressor of tumorigenicity 1, is a protein that functions as a potential tumor suppressor gene in neuroblastoma. It plays a critical role in preventing cells from entering the final stage (G1/S) of the transformation process, thereby inhibiting uncontrolled cell proliferation . Research has demonstrated that NBL1 can effectively inhibit platelet-derived growth factor-BB (PDGF-BB)-induced proliferation in human pulmonary arterial smooth muscle cells (PASMCs), suggesting its potential role in preventing pulmonary arterial remodeling in pulmonary arterial hypertension (PAH) .

What applications are NBL1 antibodies validated for in research contexts?

Commercial NBL1 antibodies, such as the rabbit recombinant monoclonal antibody [EPR12397], have been validated for several research applications including Western Blot (WB), Immunocytochemistry/Immunofluorescence (ICC/IF), and intracellular Flow Cytometry (Flow Cyt) . These applications enable researchers to detect NBL1 protein expression, visualize its cellular localization, and quantify its presence in different cell populations, respectively. The antibody has been specifically validated for use with human samples, with predicted band sizes of approximately 149 kDa and 19 kDa when analyzed by Western blot .

How does NBL1 inhibit cell proliferation at the molecular level?

NBL1 inhibits cell proliferation through multiple interconnected mechanisms at the molecular level. Studies have shown that NBL1 suppresses the formation of cyclin D1-CDK4 complexes, which are critical for cell cycle progression. Additionally, NBL1 decreases the phosphorylation of p27, a cell cycle inhibitor, thereby stabilizing p27 and promoting its growth inhibitory effects . At the signaling level, NBL1 blocks the PDGF receptor β (PDGFRβ)-p38 mitogen-activated protein kinase (MAPK) pathway, which is an upstream regulator of the aforementioned cell cycle components . This multi-level intervention in proliferative signaling explains NBL1's potent growth inhibitory effects in certain cell types.

What are the optimal experimental conditions for detecting NBL1 using immunoblotting techniques?

For optimal detection of NBL1 by Western blot, researchers should consider the following parameters based on validated protocols: use the anti-NBL1 antibody at a 1/1000 dilution, load approximately 10 μg of total protein per lane, and be prepared to detect bands at both 149 kDa and 19 kDa, which represent different forms of the protein . When preparing samples, standard cell lysis buffers containing protease inhibitors are recommended to prevent protein degradation. For visualization, both chemiluminescent and fluorescent secondary detection methods are suitable. Positive control samples such as A431 cell lysate or human prostate lysate have been validated to express detectable levels of NBL1 and should be included in experiments to confirm antibody performance .

How should researchers design experiments to study NBL1's effect on cell proliferation?

For PDGF-BB-induced proliferation models, researchers should consider dose-dependent experiments with NBL1 concentrations ranging from 0.25 to 1 μM, with 0.5 μM representing an effective concentration in published studies . Time-course experiments should include both early (3 hours) and later (24 hours) timepoints to capture both signaling events and phenotypic outcomes .

What controls are essential when studying NBL1's role in signaling pathway regulation?

When investigating NBL1's role in signaling pathway regulation, several essential controls should be included:

• Positive controls: Cells treated with established pathway activators (e.g., PDGF-BB at 10 ng/ml for PDGFRβ-p38MAPK pathway activation)

• Negative controls: Untreated cells to establish baseline signaling levels

• Pathway inhibitor controls: Cells treated with specific inhibitors (e.g., SB203580 for p38MAPK inhibition) to validate pathway specificity

• Protein knockdown controls: siRNA-mediated knockdown of key pathway components (e.g., p27) to confirm their involvement

• Time-course controls: Samples collected at multiple timepoints (e.g., 5, 15, 30, 60 minutes) to capture transient phosphorylation events

These controls help ensure the observed effects are specific to NBL1 intervention rather than experimental artifacts or off-target effects . Additionally, including both total and phosphorylated protein detection for key signaling molecules (PDGFRβ, ERK1/2, p38MAPK, JNK) provides crucial information about activation states versus expression levels.

How can researchers distinguish between NBL1's direct effects on proliferation versus secondary effects through other pathways?

To distinguish between direct and secondary effects of NBL1 on cell proliferation, researchers should employ temporal analysis and pathway dissection approaches. First, establish a detailed time course of events following NBL1 treatment, as direct effects typically occur earlier than secondary ones. For example, changes in PDGFRβ phosphorylation occur within minutes, while alterations in cyclin D1-CDK4 complex formation may take hours .

Second, use specific pathway inhibitors in combination with NBL1 treatment. If inhibiting a particular pathway (e.g., p38MAPK with SB203580) produces additive effects with NBL1, this suggests the pathways are distinct or partially overlapping. The published research demonstrates this approach, showing that combined NBL1 and SB203580 treatment further decreased cyclin D1-CDK4 activity, p27 phosphorylation, and cell proliferation compared to either treatment alone .

Third, employ protein interaction studies (co-immunoprecipitation, proximity ligation assays) to identify direct binding partners of NBL1. This can help establish which proteins NBL1 directly interacts with versus which are affected downstream through signaling cascades.

What methodological approaches can resolve contradictory findings about NBL1 expression patterns in different tissues?

When facing contradictory findings regarding NBL1 expression patterns across different tissues or disease states, researchers should implement a systematic multi-technique approach:

• Employ multiple antibodies targeting different epitopes of NBL1 to confirm specificity

• Utilize complementary detection methods beyond immunodetection, such as:

  • mRNA quantification (qRT-PCR, RNA-seq)

  • In situ hybridization to visualize transcript localization

  • Mass spectrometry-based protein identification

• Include comprehensive tissue panels with appropriate controls

• Apply stringent statistical analysis with adequate sample sizes

• Consider developmental timing and disease progression, as NBL1 expression may be dynamic

Research has shown that NBL1 is highly expressed in normal rat lung tissue but shows low expression in pulmonary arterial hypertension models . These contradictions may reflect tissue-specific regulation, disease-specific alterations, or methodological differences in detection sensitivity. Researchers should also consider potential post-translational modifications that might affect antibody recognition without altering total protein levels.

How can researchers effectively analyze the interaction between NBL1 and cell cycle regulatory proteins?

To effectively analyze interactions between NBL1 and cell cycle regulatory proteins, researchers should employ a combination of biochemical, cellular, and functional approaches:

• Co-immunoprecipitation (co-IP): This technique has successfully demonstrated reduced cyclin D1-CDK4 complex formation following NBL1 treatment . For optimal results, researchers should:

  • Use antibodies specific for either cyclin D1 or CDK4 for the pull-down

  • Perform reciprocal co-IPs to confirm interactions

  • Include appropriate negative controls (IgG, irrelevant proteins)

• Phosphorylation status analysis: Using phospho-specific antibodies (e.g., anti-phospho-p27 at Thr198) to track modifications of cell cycle regulators . This approach revealed that NBL1 decreases p27 phosphorylation, thereby increasing its stability.

• Functional validation through gene knockdown: siRNA-mediated knockdown of key proteins (e.g., p27) can confirm their functional relevance in NBL1-mediated growth suppression .

• Kinase activity assays: To directly measure the effect of NBL1 on CDK4 activity using specific substrates.

• Cell synchronization experiments: To determine at which specific cell cycle phase NBL1 exerts its effects.

These complementary approaches provide a comprehensive understanding of how NBL1 interfaces with the cell cycle machinery to inhibit proliferation.

What are the common technical challenges when using NBL1 antibodies and how can they be overcome?

When working with NBL1 antibodies, researchers may encounter several technical challenges that can be addressed with specific optimization strategies:

• Multiple band detection: NBL1 antibodies may detect bands at both 149 kDa and 19 kDa . To confirm specificity:

  • Include positive control samples (A431 cell lysate, human prostate lysate)

  • Perform antibody validation with knockdown/knockout samples

  • Use gradient gels to better resolve protein bands

• Variable signal intensity: To enhance detection:

  • Optimize antibody concentration (starting with 1/1000 dilution for Western blot, 1/100 for ICC/IF, and 1/10 for flow cytometry)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Test different blocking agents (BSA vs. non-fat milk)

  • Consider signal amplification systems for low-abundance targets

• Non-specific binding: To reduce background:

  • Increase washing duration and frequency

  • Optimize blocking conditions

  • Pre-absorb antibodies with relevant tissues/cell lysates

  • Use monoclonal antibodies like EPR12397 for higher specificity

• Inconsistent results across applications: Different applications may require distinct optimization strategies:

  • For Western blot: optimize protein transfer conditions

  • For ICC/IF: test different fixation methods (paraformaldehyde vs. methanol)

  • For flow cytometry: carefully optimize permeabilization protocols for intracellular detection

How can researchers optimize cell-based assays to study NBL1's effect on the PDGF signaling pathway?

To optimize cell-based assays for studying NBL1's effects on PDGF signaling, researchers should consider:

• Cell model selection: Human PASMCs have been successfully used to study NBL1 effects on PDGF signaling . Consider:

  • Primary cells vs. cell lines (primary cells better reflect physiological responses)

  • Cell passage number (use low passage cells for consistent responses)

  • Growth conditions (serum starvation before stimulation to reduce baseline signaling)

• PDGF-BB stimulation parameters:

  • Concentration: 10 ng/ml PDGF-BB has been validated for inducing robust responses

  • Time course: Examine both early (minutes) for signaling events and later (24 hours) for proliferative responses

  • Pre-treatment vs. co-treatment with NBL1 (pre-treatment of 1-2 hours recommended)

• Signaling readouts:

  • Phosphorylation-specific antibodies to detect activated signaling components (p-PDGFRβ, p-p38MAPK, p-ERK1/2)

  • Multiple timepoints to capture transient phosphorylation events

  • Both Western blot and cell-based assays (ELISA, high-content imaging) for comprehensive analysis

• Pathway inhibitor controls:

  • Include specific inhibitors like SB203580 (p38MAPK inhibitor) to confirm pathway involvement

  • Use inhibitors at validated concentrations to avoid off-target effects

  • Consider combination treatments to identify additive or synergistic effects

By systematically optimizing these parameters, researchers can develop robust assays to characterize NBL1's effects on PDGF signaling with high reproducibility and physiological relevance.

What quantitative methods are most appropriate for analyzing NBL1's effects on cell cycle progression?

To quantitatively analyze NBL1's effects on cell cycle progression, researchers should employ complementary methods that capture different aspects of the cell cycle:

• Flow cytometry-based cell cycle analysis:

  • DNA content analysis using propidium iodide staining

  • BrdU incorporation to measure S-phase entry

  • Dual parameter analysis (e.g., DNA content + cyclin expression) to precisely define cell cycle phases

  • Quantification of cell populations in G0/G1, S, and G2/M phases

• Proliferation marker analysis:

  • Quantitative Western blot or immunofluorescence for PCNA expression

  • EdU incorporation assay for direct measurement of DNA synthesis

  • Ki-67 staining for identifying cycling cells

• Cell cycle protein dynamics:

  • Quantitative analysis of cyclin D1-CDK4 complex formation via co-immunoprecipitation

  • Phosphorylation status of p27 at specific residues (e.g., Thr198)

  • Expression levels of cell cycle regulators (cyclins, CDKs, CDK inhibitors)

• Real-time cell cycle progression:

  • Live-cell imaging with fluorescent cell cycle indicators

  • FUCCI (Fluorescence Ubiquitination Cell Cycle Indicator) system for real-time visualization

• Mathematical modeling:

  • Apply computational models to integrate multiple measurements

  • Determine rate constants for cell cycle phase transitions

  • Predict the impact of NBL1 on specific cell cycle checkpoints

For optimal results, researchers should combine at least 2-3 of these approaches to build a comprehensive picture of how NBL1 affects cell cycle dynamics. Statistical analysis should include appropriate tests for distribution comparison and time-course data analysis.

How might NBL1's tumor suppressor activities be exploited in cancer research beyond neuroblastoma?

NBL1's demonstrated tumor suppressor activities present opportunities for broader applications in cancer research beyond neuroblastoma. Based on its mechanistic actions, several promising research directions emerge:

• Pulmonary vascular diseases: Given NBL1's inhibitory effects on PDGF-BB-induced PASMC proliferation, investigating its potential therapeutic role in pulmonary arterial hypertension (PAH) and other vascular remodeling diseases is warranted . Researchers could explore:

  • NBL1 expression patterns across various pulmonary vascular diseases

  • Development of NBL1-based therapies to inhibit vascular smooth muscle cell proliferation

  • Combined approaches targeting multiple aspects of vascular remodeling

• Cell cycle-driven cancers: NBL1's ability to inhibit cyclin D1-CDK4 activity and stabilize p27 makes it potentially relevant for cancers characterized by dysregulated cell cycle control . Investigations could focus on:

  • Screening cancer types for NBL1 expression correlation with prognosis

  • Examining NBL1's effects on cancer stem cell self-renewal

  • Developing combination approaches with established CDK4/6 inhibitors

• PDGF-dependent tumor types: Since NBL1 blocks PDGFRβ signaling, it may have relevance in PDGF-driven tumors such as certain gliomas, gastrointestinal stromal tumors, and dermatofibrosarcoma protuberans. Research could examine:

  • NBL1's efficacy in inhibiting growth of PDGF-dependent tumor models

  • Potential synergy with existing tyrosine kinase inhibitors targeting PDGF receptors

  • Development of tumor-targeted NBL1 delivery systems

These approaches require careful experimental design, including appropriate in vitro and in vivo models, pathway analysis, and correlation with clinical samples to establish translational relevance.

What methodological approaches are most effective for studying NBL1's role in regulating p38MAPK signaling?

To effectively study NBL1's role in regulating p38MAPK signaling, researchers should implement a comprehensive methodological toolkit:

• Phosphorylation-specific detection methods:

  • Western blotting with phospho-specific antibodies for p38MAPK activation (phospho-p38)

  • Phospho-flow cytometry for single-cell resolution of p38MAPK activation

  • Phospho-proteomics to identify all phosphorylation changes in the pathway

  • In-cell Western or ELISA-based phosphorylation assays for higher throughput

• Temporal analysis approaches:

  • Detailed time-course experiments (5, 15, 30, 60 minutes, etc.) to capture activation kinetics

  • Pulse-chase experiments to determine pathway persistence

  • Live-cell reporters for real-time monitoring of p38MAPK activity

• Pathway perturbation strategies:

  • Specific inhibitors like SB203580 to block p38MAPK activity

  • siRNA/shRNA knockdown of pathway components

  • CRISPR/Cas9 knockout of key signaling nodes

  • Expression of constitutively active or dominant-negative p38MAPK constructs

• Downstream target analysis:

  • Activity assays for p38MAPK substrates

  • Gene expression profiling following NBL1 treatment

  • Chromatin immunoprecipitation to identify transcriptional effects

• Protein interaction studies:

  • Co-immunoprecipitation to detect protein complexes

  • Proximity ligation assay for in situ detection of protein interactions

  • FRET/BRET approaches for real-time interaction monitoring

The research has already established that NBL1 reduces p38MAPK phosphorylation induced by PDGF-BB in human PASMCs . These methodological approaches would further elucidate the molecular mechanisms and identify additional components in this signaling axis.

How should researchers design experiments to investigate potential contradictions between NBL1's effects in different cellular contexts?

When investigating potential contradictions in NBL1's effects across different cellular contexts, researchers should implement a systematic experimental design that accounts for biological variability and mechanistic differences:

• Standardized comparative analysis:

  • Simultaneously test multiple cell types under identical experimental conditions

  • Include primary cells and established cell lines from relevant tissues

  • Apply consistent treatment protocols (concentrations, timing, media conditions)

  • Use the same batch of reagents and detection methods across experiments

• Context-dependent variable identification:

  • Perform baseline characterization of each cellular system:

    • Receptor expression profiling (PDGFRβ levels and activation status)

    • Cell cycle regulator expression (p27, cyclins, CDKs)

    • Pathway component expression (MAPK family members)

  • Conduct gene expression profiling to identify key differences between responsive and non-responsive contexts

• Mechanistic dissection approaches:

  • Employing genetic manipulation to introduce or remove specific components:

    • Transfer of missing components to non-responsive systems

    • Knockdown of potential inhibitory factors

  • Pathway reconstruction in simplified systems

  • Domain mapping to identify critical regions of NBL1 for different functions

• Physiological relevance assessment:

  • Correlate in vitro findings with in vivo observations

  • Analyze tissue-specific expression patterns in normal versus disease states

  • Consider developmental and pathological context

This structured approach can help reconcile apparent contradictions, such as why NBL1 might strongly inhibit proliferation in PASMCs but show different effects in other cell types, or why its expression varies between normal lung tissue and PAH models . By identifying the critical determinants of NBL1 responsiveness, researchers can develop more targeted approaches for specific disease contexts.