CEND1 Antibody

What is CEND1 and why is it significant in neuroscience research?

CEND1 (Cell Cycle Exit and Neuronal Differentiation 1) is a neuronal lineage-specific modulator involved in synchronizing cell cycle exit and differentiation of neuronal precursors. It plays a critical role in neuronal development by promoting the exit of neural progenitor cells from the cell cycle and initiating their differentiation into mature neurons . CEND1 is expressed throughout the neuronal lineage, from neural stem/progenitor cells to mature neurons, and is associated with neuron-generating cell divisions . Its significance stems from its involvement in coordinating cell cycle progression/exit and differentiation, which is essential for generating appropriate numbers of neurons and forming correct neuronal circuits in the adult brain .

What are the key characteristics of CEND1 protein structure that researchers should consider when selecting antibodies?

CEND1 protein contains a proline-rich signaling domain with several PXXP repeats that represent putative SH3-binding sites involved in protein-protein interactions . Researchers should select antibodies that can recognize specific epitopes, particularly noting that:

• The C-terminal region (specifically the last three amino acids residues 147-149, RKK) mediates CEND1's outer-membrane mitochondrial localization

• The N-terminal region contains important sequence elements used for some commercially available antibodies

• CEND1 has a predicted molecular weight of approximately 15 kDa, but often appears at 22 kDa in Western blots

When selecting antibodies, researchers should verify which region the antibody targets and whether this might be affected by potential post-translational modifications or protein-protein interactions.

What species reactivity should be considered when selecting CEND1 antibodies?

Based on available commercial antibodies, researchers should consider the following species reactivity patterns:

When planning cross-species studies, researchers should carefully evaluate antibody validation data for each species of interest.

What are the optimal working dilutions for CEND1 antibodies in different experimental applications?

Based on validated protocols from multiple sources, recommended dilutions vary by application:

Researchers should always optimize dilutions for their specific experimental conditions and sample types.

What antigen retrieval methods are recommended for CEND1 immunohistochemistry?

Heat-mediated antigen retrieval is strongly recommended before commencing with IHC staining protocols for CEND1 . This is particularly important when working with paraffin-embedded tissues, as CEND1 epitopes may be masked during fixation and embedding processes. The recommended protocol involves:

• Deparaffinization and rehydration of tissue sections

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking in 3% bull serum albumin for 30 minutes

• Overnight incubation with primary antibody at 4°C

• Incubation with appropriate secondary antibody (e.g., Goat anti-rabbit IgG)

For frozen sections, antigen retrieval may be less critical, but optimization is still recommended.

How can I validate the specificity of my CEND1 antibody?

Validating CEND1 antibody specificity is crucial for reliable research results. Recommended validation approaches include:

• Positive and negative tissue controls: Human brain tissue shows strong CEND1 expression, while non-neuronal tissues typically show minimal expression

• Cell line controls:

  • Positive controls: U-87 MG, Neuro-2a, and SH-SY5Y cell lines

  • Relative expression comparison: HEB cells (lower expression) vs. neuronal cells (higher expression)

• Knockdown/knockout validation: CEND1 siRNA or CEND1 knockout models provide the most stringent specificity control

• Western blot profile: Confirm the expected molecular weight (predicted 15 kDa, often observed at 22 kDa)

• Subcellular localization pattern: CEND1 is primarily localized to mitochondria, particularly the outer membrane

What is the subcellular localization of CEND1 and how should this inform immunostaining protocols?

CEND1 has been definitively identified as a neuronal mitochondrial protein that predominantly localizes to the outer mitochondrial membrane . Specifically:

• Subcellular fractionation and immunofluorescence staining reveal that CEND1 is primarily mitochondrial

• Trypsin digestion experiments of intact mitochondria demonstrate that CEND1 is digested without Triton X-100 treatment, confirming its outer membrane localization

• The last three amino acids (147-149, RKK) at the C-terminus mediate this outer-membrane localization; deletion or mutation of this sequence prevents mitochondrial localization

For immunostaining protocols, researchers should:

• Use mitochondrial markers (like MFN2 or COX IV) as co-staining controls

• Ensure fixation methods preserve mitochondrial integrity

• Consider permeabilization steps carefully to maintain outer membrane proteins

• Use confocal microscopy to confirm co-localization with mitochondrial markers

How does CEND1 expression vary across brain regions and developmental stages?

CEND1 expression patterns vary significantly across development and brain regions:

• Developmental regulation:

  • CEND1 is expressed throughout the neuronal lineage from neural stem/progenitor cells to mature neurons

  • Expression increases during the transition from proliferating neural precursors to post-mitotic neurons

  • CEND1 mRNA levels are significantly higher in P7 mouse hearts compared to P1, correlating with reduced cardiomyocyte proliferation

• Brain region specificity:

  • CEND1 plays significant roles in cerebellar cortex development

  • It is critical for proper development of GABAergic interneurons in the cerebral cortex, hippocampus, and amygdala

  • CEND1 knockout mice show abnormalities particularly in Purkinje cell differentiation

• Pathological changes:

  • CEND1 expression is significantly reduced in glioma tissues compared to normal brain tissue

  • CEND1 decreases significantly with the progression of Alzheimer's disease in mouse models (APP/PS1 and 5xFAD)

  • After traumatic brain injury, CEND1 levels are downregulated in mouse brain tissue

How is CEND1 expression altered in neurological disease models and what methodological considerations are important?

CEND1 expression shows significant changes in multiple neurological disease models:

• Alzheimer's Disease (AD):

  • CEND1 protein levels are significantly decreased in hippocampus of 10-month-old APP/PS1 mice compared to age-matched controls

  • In 5xFAD mice, CEND1 progressively decreases with disease progression (from 2 to 9 months)

  • In cellular models, both Aβ(1-42) treatment of primary neurons and APP695 overexpression in N2a cells dramatically decrease CEND1 expression

  • Methodological consideration: Use age-matched controls and track expression across disease progression

• Glioma:

  • CEND1 expression is significantly reduced in glioma tissues and cell lines compared to normal brain tissue

  • Low CEND1 expression correlates with shorter survival of glioma patients

  • CEND1 knockdown promotes glioma cell growth, migration, invasion, and chemoresistance to temozolomide (TMZ)

  • Methodological consideration: Compare expression across different glioma grades and treatment-resistant variants

• Traumatic Brain Injury (TBI):

  • Quantitative proteomic analysis shows CEND1 downregulation in mouse brain tissue after TBI

  • Transplantation of CEND1-transfected neural stem cells improves outcomes after TBI

  • Methodological consideration: Temporal analysis of expression changes after injury is critical

• Zika Virus Infection:

  • CEND1 interacts specifically with the nonstructural viral protein NS4B

  • CEND1 knockdown results in inhibition of Zika virus replication

  • Methodological consideration: Protein-protein interaction studies require validated antibodies for co-immunoprecipitation

What are the major signaling pathways associated with CEND1 and how can antibodies be used to study these interactions?

CEND1 operates through several key signaling pathways that can be studied using antibody-based techniques:

• p53/Cyclin D1/pRb Pathway:

  • CEND1 activates the p53-pRb signaling pathway controlling the balance between cell proliferation and cell cycle exit

  • CEND1's antiproliferative effect is associated with Cyclin D1 downregulation and cytoplasmic relocation

  • Research approach: Combined immunofluorescence to track subcellular localization of Cyclin D1 with CEND1 expression

• Notch Signaling Pathway:

  • CEND1 promotes neuronal differentiation by suppressing Notch1 signaling

  • CEND1 overexpression leads to downregulation of Notch1, Hes5, Olig2, Cash1, and Pax7

  • Research approach: ChIP assays using CEND1 antibodies to identify potential regulatory regions

• NF-κB Pathway:

  • CEND1 inhibits glioma cell proliferation and resistance to temozolomide by inhibiting the NF-κB pathway

  • CEND1 knockdown facilitates p-p65 expression, while CEND1 overexpression suppresses p-p65 expression

  • Research approach: Co-immunoprecipitation studies to detect interaction partners

• CDK5-CEND1-Drp1 Axis:

  • CDK5/p25 binds to and phosphorylates CEND1 at serine 10, promoting CEND1 degradation

  • CEND1 depletion induces mitochondrial fission dysfunctions by upregulation of Drp1

  • Research approach: Phospho-specific antibodies or phospho-tag gels to detect CEND1 phosphorylation states

How can CEND1 antibodies be used in studies of protein-protein interactions and what technical challenges should be anticipated?

CEND1 interacts with multiple proteins, and studying these interactions requires sophisticated antibody-based approaches:

• Known interaction partners:

  • RanBPM: A scaffolding protein in the nervous and immune systems

  • Ahi1/Jouberin: Implicated in Joubert syndrome

  • Zika virus NS4B protein

  • Dyrk1B: Forms a tripartite interaction with RanBPM and CEND1

  • CDK5/p25: Binds and phosphorylates CEND1

• Technical approaches and considerations:

  • Co-immunoprecipitation: Use CEND1 antibodies to pull down protein complexes

    • Challenge: CEND1's small size (15 kDa) may make it difficult to distinguish from antibody light chains

    • Solution: Use antibodies directly conjugated to beads or cross-linked to protein A/G

  • Proximity Ligation Assay (PLA):

    • Challenge: Requires highly specific antibodies raised in different species

    • Solution: Combine rabbit monoclonal anti-CEND1 with mouse antibodies against interaction partners

  • FRET/BRET analysis:

    • Challenge: Requires epitope tagging that might disrupt mitochondrial localization

    • Solution: Careful positioning of tags to avoid the C-terminal mitochondrial targeting sequence

  • Mass spectrometry identification:

    • Challenge: CEND1 antibodies may have varying efficiencies in different buffer conditions

    • Solution: Optimize immunoprecipitation conditions specifically for mass spectrometry compatibility

What are the critical considerations when using CEND1 antibodies in functional reprogramming studies?

CEND1 has shown promising results in neuronal reprogramming applications, with several important antibody-related considerations:

• Monitoring CEND1 expression during reprogramming:

  • CEND1 has been used successfully in direct reprogramming of mouse astrocytes to functional neurons

  • Researchers should track CEND1 expression temporally during the reprogramming process

  • Use of multiple antibodies targeting different epitopes may provide more comprehensive information on protein functionality

• Evaluating downstream effects:

  • CEND1 overexpression leads to downregulation of pluripotency marker Oct3 and upregulation of proneural genes Neurogenin 1 and Mash1

  • Antibodies for these markers should be included in multiplexed analysis

  • Co-staining with markers for cell cycle (Ki67), neuronal differentiation (TuJ1, MAP2), and GABAergic fate (GAD65/67) is recommended

• Functional validation:

  • CEND1-overexpressing neural stem cells tend to adopt a GABAergic phenotype after transplantation

  • Antibodies specific for GABAergic interneuron markers should be included in analysis panels

  • Combined electrophysiological recording with immunocytochemistry provides the most comprehensive functional assessment

• Transplantation studies:

  • CEND1-overexpressing grafts attenuate astrogliosis after injury

  • Dual immunohistochemistry for CEND1 and GFAP (astrocyte marker) can quantify this effect

  • Careful optimization of antigen retrieval and antibody penetration in thick tissue sections is essential

What challenges might researchers encounter when studying post-translational modifications of CEND1?

CEND1 undergoes important post-translational modifications that affect its function, presenting specific challenges for antibody-based detection:

• Phosphorylation:

  • CDK5/p25 phosphorylates CEND1 at serine 10, promoting its degradation

  • Challenge: Standard antibodies may not distinguish between phosphorylated and non-phosphorylated forms

  • Solution: Use phospho-specific antibodies when available, or phospho-tag SDS-PAGE followed by Western blotting

• Protein degradation:

  • CEND1 degradation is regulated in physiological and pathological conditions

  • Challenge: Degradation products may not be detected by antibodies targeting specific epitopes

  • Solution: Use antibodies targeting different regions of CEND1 and optimize sample preparation to minimize degradation

• Protein-protein interactions affecting epitope accessibility:

  • CEND1's interactions with numerous partners may mask epitopes

  • Challenge: False negatives in immunostaining or Western blotting

  • Solution: Test multiple antibodies targeting different epitopes and optimize sample preparation conditions

• Mitochondrial localization:

  • The C-terminal RKK motif is critical for mitochondrial targeting

  • Challenge: Modifications affecting this region may alter localization without changing expression

  • Solution: Combined subcellular fractionation with Western blotting to assess distribution changes