NEURL1 Antibody

What is the optimal antibody dilution for detecting endogenous NEURL1 in Western blots?

• Using higher antibody concentrations (1:500) for initial experiments

• Extending membrane exposure times to 5-10 minutes

• Loading at least 30-50μg of total protein per lane

• Employing enhanced chemiluminescence detection systems

Remember that fresh preparation of lysates significantly improves detection quality compared to frozen samples.

Which tissues show highest endogenous expression of NEURL1 for positive controls?

NEURL1 shows tissue-specific expression patterns that should be considered when selecting positive controls:

Notably, NEURL1 expression is significantly reduced in medulloblastoma compared to normal cerebellar tissue, with particularly low levels in hedgehog-activated tumors . For immunohistochemistry studies, mouse brain tissue sections work exceptionally well as positive controls, showing distinct staining patterns when using heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) .

What are the key structural domains to consider when selecting NEURL1 antibodies?

NEURL1 contains several functional domains that should guide antibody selection based on experimental goals:

• NHR domains (Neuralized Homology Regions): Mediate protein-protein interactions, particularly with substrates like PDE9A

• RING domain: Essential for E3 ubiquitin ligase activity

When selecting antibodies:

• Antibodies targeting the center region (amino acids 158-186) provide robust detection of full-length NEURL1

• Antibodies against NHR domains are valuable for studying protein-protein interactions

• RING domain-targeting antibodies may interfere with ubiquitination activity in functional assays

For investigating specific protein interactions (e.g., with PDE9A or Jagged1), avoid antibodies that target the interaction interfaces within the NHR domains .

How should researchers design co-immunoprecipitation experiments to study NEURL1 interactions?

Successful co-immunoprecipitation of NEURL1 and its binding partners requires careful experimental design:

• Cell system selection: HEK293-FT cells are recommended due to endogenous expression of both NEURL1 and interaction partners like PDE9A

• Tagging strategies:

  • Use both N- and C-terminally tagged constructs to minimize tag-specific effects

  • Validated tags include Flag for NEURL1/NEURL1B and V5His for interaction partners

  • Verify that tags don't interfere with protein-protein interactions

• Immunoprecipitation protocol optimization:

  • Lyse cells in buffer containing 150mM NaCl, 1% Triton X-100, 50mM Tris-HCl (pH 7.4), and protease inhibitors

  • Pre-clear lysates with protein A/G beads for 1 hour

  • Incubate with 2-5μg antibody overnight at 4°C

  • Add fresh protein A/G beads for 1-2 hours before washing

• Controls:

  • Include IgG control immunoprecipitations

  • Test both forward and reverse co-IPs to confirm interactions

  • Include domain mutants to map interaction regions

Studies have successfully demonstrated NEURL1 interactions with PDE9A using this approach, showing that both NHR domains independently, but not the RING domain, mediate this interaction .

What methodological approaches best reveal NEURL1's E3 ligase activity toward specific substrates?

Investigating NEURL1's E3 ligase activity requires multiple complementary approaches:

• Ubiquitination assays:

  • Co-express NEURL1, substrate (e.g., PDE9A or Jagged1), and tagged ubiquitin in HEK293 cells

  • Use wild-type ubiquitin alongside K0 (all lysines mutated) and K-only mutants to determine ubiquitin chain linkage types

  • For NEURL1-mediated degradation of PDE9A, the K27 ubiquitin mutant shows significant effects

  • For Jagged1, evidence suggests monoubiquitination rather than polyubiquitination

• Protein stability analysis:

  • Perform cycloheximide chase assays (80μg/mL) with timepoints at 0, 2, 4, 6, and 8 hours

  • Compare substrate degradation rates with and without NEURL1 expression

  • Include proteasome inhibitor (MG132, 10μM) or lysosome inhibitor (NH₄Cl, 20μM) controls

• Endocytosis assessment for Notch ligands:

  • Immunofluorescence microscopy to track Jagged1 endocytosis

  • Co-localization analysis with endocytic markers

  • Quantification of surface versus internalized Jagged1

Research has demonstrated that NEURL1 promotes ubiquitination and subsequent degradation of PDE9A via K27-linked ubiquitin chains , while also mediating Jagged1 endocytosis through monoubiquitination .

How do I address contradictory data regarding NEURL1's role in Notch signaling regulation?

The literature contains seemingly contradictory findings regarding NEURL1's role in Notch signaling:

To resolve these contradictions, design experiments that:

• Establish cellular context:

  • Perform comparative studies across neural progenitors, differentiated neurons, and tumor cells

  • Measure baseline Notch activity in each system

• Assess direct vs. indirect effects:

  • Use time-course experiments after NEURL1 manipulation

  • Apply specific Notch pathway inhibitors (γ-secretase inhibitors) alongside NEURL1 manipulation

  • Employ NEURL1 constructs with mutations in different functional domains

• Examine interactions with regulatory factors:

  • Test how neuritin affects NEURL1 function across different cell types

  • Investigate interactions with other Notch pathway components

• Measure comprehensive pathway outputs:

  • Assess Jagged1 endocytosis, NICD generation, and target gene expression simultaneously

  • Use ChIP assays to examine NICD recruitment to target promoters

Evidence suggests that NEURL1 typically promotes Notch signaling through ligand endocytosis, but this effect can be modulated by factors like neuritin, which promotes NEURL1 degradation via the 26S proteasome and weakens NEURL1's affinity for Jagged1 .

What are the most effective antibody validation strategies for NEURL1 detection?

Rigorous validation is critical for NEURL1 antibodies due to detection challenges:

• Multiple validation approaches:

  • Overexpression controls: Compare untransfected vs. NEURL1-transfected cell lysates

  • Gene silencing: Use siRNA knockdown (neuritin siRNA-3 shows highest efficiency )

  • Peptide competition: Block with the immunizing peptide (shows clear band elimination )

  • Cross-reactivity testing: Ensure specificity using multiple cell/tissue sources

• Commercial antibody limitations:

  • Several commercial antibodies fail to detect endogenous NEURL1

  • Custom-raised polyclonal antibodies show superior specificity and sensitivity

  • Consider developing your own antibodies for critical applications

• Recommended validation protocol:

  • Test antibody on Western blots of brain tissue (high endogenous expression)

  • Include molecular weight markers to confirm the 62kD band

  • Use transfected cells expressing tagged NEURL1 as positive controls

  • Perform peptide competition assays to confirm specificity

When troubleshooting detection issues, remember that NEURL1 expression is tissue-specific and often low in cell lines, requiring optimization of protein extraction and detection methods.

How can I optimize immunofluorescence protocols for detecting subcellular localization of NEURL1?

NEURL1 shows distinct subcellular localization patterns that can be challenging to detect:

• Fixation optimization:

  • -20°C methanol fixation works well for MCF-7 cells

  • 4% paraformaldehyde (10 minutes) followed by 0.1% Triton X-100 permeabilization (5 minutes) for neuronal cells

  • Avoid overfixation which can mask epitopes

• Subcellular localization patterns to expect:

  • Cytoplasm with perinuclear concentration

  • Cell membrane (peripheral)

  • Dendrites in neuronal cells

  • Postsynaptic density in neurons (co-localizes with PSD95)

• Co-localization markers:

  • For dendritic localization: MAP2

  • For synaptic localization: PSD95

  • For membrane trafficking: JAG1

  • For protein interactions: PDE9A or neuritin

• Signal amplification strategies:

  • Use high-sensitivity detection systems like tyramide signal amplification

  • Consider confocal microscopy for better resolution of subcellular structures

  • Apply deconvolution algorithms to improve signal-to-noise ratio

Researchers should note that NEURL1 shows co-localization with JAG1 at the cell surface and with CPEB3 at apical dendrites of CA1 neurons , providing valuable positive controls for localization studies.

What technical challenges arise when studying NEURL1 as a tumor suppressor in cancer models?

Investigating NEURL1's tumor suppressor role presents several methodological challenges:

• Expression level considerations:

  • NEURL1 expression is significantly reduced in medulloblastoma (90% of tumors show <50% of normal expression)

  • Particularly low in hedgehog-activated tumors

  • Maximum expression in cell lines is ~1.4% of normal cerebellar tissue

• Mechanisms of downregulation:

  • Histone modifications appear to be the primary mechanism

  • 10q loss, sequence mutations, and promoter hypermethylation play minor roles

  • Design experiments to test multiple epigenetic mechanisms simultaneously

• Functional assays:

  • Colony formation assays show decreased colony-forming ability with NEURL1 expression

  • Tumor sphere formation assays reveal inhibition with NEURL1 restoration

  • Xenograft models demonstrate reduced growth with NEURL1 expression

  • Cell cycle analysis reveals increased apoptosis in NEURL1-expressing cells

• Integration with Notch pathway analysis:

  • Measure effects on Jagged1 expression

  • Assess target genes HES1 and HEY1

  • Develop rescue experiments using NICD expression

When designing these experiments, researchers should include comprehensive controls and perform time-course studies to distinguish direct effects from secondary consequences of NEURL1 expression.

How can researchers effectively study the relationship between neuritin and NEURL1 in neural development?

The interaction between neuritin and NEURL1 represents an emerging area requiring specific methodological approaches:

• Interaction characterization:

  • Yeast two-hybrid screens identify neuritin as a NEURL1 interactor

  • Confirm with forward and reverse co-IP in mammalian cells like 293T

  • Test domain-specific interactions using truncation constructs

• Functional relationship studies:

  • Neuritin suppresses NEURL1 levels and promotes its degradation via 26S proteasome

  • Neuritin weakens NEURL1-Jagged1 affinity in a dose-dependent manner

  • Test varying concentrations (40ng vs. 80ng neuritin plasmid) to observe dose effects

• Consequences for Notch signaling:

  • Neuritin reduces JAG1 endocytosis promoted by NEURL1

  • This leads to decreased NICD and HES1 levels

  • Design rescue experiments with NEURL1 overexpression to confirm specificity

• Developmental context:

  • Examine expression patterns during neural development stages

  • Investigate consequences for neurite growth and neuronal differentiation

  • Consider neuritin as a potential negative regulator of NEURL1 that inhibits Notch signaling to promote neurite growth

These studies reveal that neuritin functions as an upstream and negative regulator of NEURL1, inhibiting Notch signaling and potentially promoting neural development through this mechanism .

What methodological approaches best illuminate the dynamics of NEURL1-mediated protein degradation?

Studying NEURL1's role in protein degradation requires sophisticated approaches:

• Protein half-life determination:

  • Cycloheximide chase assays with 80μg/mL concentration

  • Sample collection at 0, 2, 4, 6, and 8 hours

  • Western blot analysis with densitometry quantification

  • Compare degradation rates with and without NEURL1 expression

• Degradation pathway identification:

  • Proteasome inhibition: MG132 (10μM, 4 hours before harvesting)

  • Lysosomal inhibition: NH₄Cl (20μM, 4 hours before harvesting)

  • Compare protein levels to determine primary degradation route

• Ubiquitin chain linkage analysis:

  • Co-express NEURL1 with wild-type ubiquitin, K0 ubiquitin, or K-only mutants

  • For PDE9A, K27-linked ubiquitin chains appear critical for degradation

  • For neuritin-NEURL1 interaction, study how neuritin affects NEURL1 ubiquitination

• Binding kinetics assessment:

  • Determine optimal time for substrate binding (e.g., 24h for Jagged1-NEURL1)

  • Test how regulatory factors like neuritin affect binding affinity over time

  • Use co-IP with increasing concentrations of competitors to measure relative affinity

These approaches have revealed that NEURL1-mediated degradation of PDE9A primarily requires K27 ubiquitin linkages , while neuritin can regulate NEURL1 activity by promoting its degradation through the 26S proteasome pathway .