APC7 Antibody

What is APC7 and what is its primary function in cells?

APC7 is a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). This multiprotein complex is responsible for substrate ubiquitination, marking proteins for degradation by the proteasome. APC7 contains multiple tandem TPR (tetratricopeptide repeat) motifs that often promote protein-protein interactions . Within the APC complex, APC7 plays a critical role in the stable recruitment and ubiquitination of substrates, making it essential for proper APC functioning . In structural studies, APC7 has been found to form a dimer atop the TPR lobe of the complex, contributing to its structural integrity .

How is APC7 expression regulated in different tissues?

APC7 shows tissue-specific expression patterns with notable enrichment in the brain compared to other tissues. In wild-type mice, APC7 protein is more abundant in the brain relative to other tissues . Expression analysis also demonstrates that APC7 and other APC subunits (including coactivator proteins Cdh1 and Cdc20) decline during mouse brain development . Within the brain, subcellular fractionation studies have shown that APC7 and APC3 are enriched in the nucleus relative to other APC subunits in the mouse cerebral cortex and cerebellum . Single-cell RNA sequencing of human brain tissue confirms that APC7 is expressed in both neuronal progenitors and mature neurons in the cortex and cerebellum .

What are the key considerations when selecting an APC7 antibody for research?

When selecting an APC7 antibody, researchers should consider several critical factors:

• Specificity: Verify that the antibody specifically recognizes APC7 without cross-reactivity to other APC subunits. Antibodies raised against specific epitopes, such as the carboxy terminus of human APC7, often provide better specificity . Validation can be performed by immunoblotting, where APC7-specific antibodies should recognize a distinct 63-kDa band .

• Species reactivity: Ensure the antibody recognizes APC7 from your experimental species. Some antibodies, like those described in the literature, can recognize APC7 from multiple species including human, mouse, and rat .

• Application compatibility: Confirm that the antibody works in your intended applications. Commercial antibodies are typically validated for specific applications such as Western blotting (WB), immunofluorescence (IF), and ELISA .

• Clonality: Consider whether polyclonal or monoclonal antibodies better suit your research needs. Polyclonal antibodies may provide stronger signals by recognizing multiple epitopes, but monoclonal antibodies offer greater consistency between batches .

What are the optimal protocols for immunohistochemical detection of APC7 in tissue samples?

For optimal immunohistochemical detection of APC7 in tissue samples, the following methodological approach is recommended:

• Fixation and embedding: Fix specimens in 10% buffered formaldehyde and embed in paraffin using routine methods .

• Sectioning and mounting: Cut sections 5 μm thick and place on silane-coated glass slides. Dry at 50°C for 2 hours, deparaffinize in xylene, rehydrate in graded ethanol, and wash in distilled water .

• Antigen retrieval: To optimize antigen retrieval, dip sections in citrate buffer (10 mmol/l, pH 6.0) and heat for 15 minutes in a microwave oven. This step is critical for breaking protein cross-links formed during fixation and exposing antigenic sites .

• Blocking endogenous activity: Block endogenous peroxidase activity by pretreating with 0.3% hydrogen peroxide for 10 minutes .

• Primary antibody application: After washing with 50 mmol/l Tris buffer (pH 7.5), apply anti-APC7 antibodies at a dilution of 1:50 or 1:100 and incubate overnight at 4°C .

• Secondary antibody and detection: Incubate with biotinylated secondary antibodies (1:50 dilution) for 20 minutes, followed by streptavidin peroxidase for 20 minutes, and stain with 3-amino-9-ethylcarazole. Counterstain with hematoxylin before mounting .

• Controls: Include positive and negative controls in each staining procedure to ensure staining equivalence and specificity. Use unstained tissue samples as negative controls and strongly stained tissue samples as positive controls .

How can researchers validate the specificity of APC7 antibodies?

Validating APC7 antibody specificity is crucial for obtaining reliable research results. Multiple complementary approaches should be employed:

• Immunoblotting validation: Perform Western blot analysis using cell or tissue extracts. APC7-specific antibodies should recognize a distinct 63-kDa band. Compare your results with commercially available antibodies to confirm specificity .

• Immunoprecipitation: Conduct immunoprecipitation experiments to verify that the antibody can pull down not only APC7 but also associated APC components. For example, APC7 antibodies should precipitate both APC3 and APC6 components, while APC3 antibodies should precipitate APC6 and APC7 .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate that this blocks specific binding. This is especially important for antibodies raised against peptide immunogens .

• Cross-species reactivity: Test the antibody in multiple species if your research requires cross-species comparisons. Some APC7 antibodies have been shown to recognize same-sized antigens from both mouse and human cells .

• Immunohistochemical pattern: Verify that the subcellular localization pattern matches expected distribution (primarily nuclear during interphase for APC7) .

What role does APC7 play in neuronal development and function?

APC7 serves critical functions in neuronal development and function as evidenced by both human and animal model studies:

• Essential for brain development: Mutations in the ANAPC7 gene cause an intellectual disability syndrome, demonstrating APC7's essential role in human brain development. Studies identified an 8054 bp deletion within the ANAPC7 locus that includes exons 4-6, resulting in the p.Ile171GlufsX14 allele that leads to frameshift in exon 7 and loss of the APC7 protein .

• Developmental expression patterns: APC7 is expressed in both neuronal progenitors and mature neurons. In situ RNA hybridization in P7 mouse cerebellum shows APC7 mRNA in neuronal progenitors within the external granule layer (EGL) and in differentiated neurons of the internal granule layer (IGL). APC7 expression also persists beyond the proliferative ventricular zone in the developing thalamus on embryonic day 16 (E16) and is detected in CA1 of the hippocampus on postnatal day 7 (P7) .

• Regulation of chromatin-associated proteins: APC7 is required for the function of Cdh1-APC in neurons to regulate the chromatin-associated protein Ki-67. Upon APC7 loss, deregulated neuronal Ki-67 localizes predominantly to constitutive heterochromatin, suggesting that APC7 plays a role in heterochromatin regulation in neurons .

• Ubiquitin signaling in chromatin regulation: APC7 and Cdh1-APC function in ubiquitin signaling pathways that regulate chromatin during brain development, with implications for understanding neurodevelopmental disorders of cognition .

How do researchers investigate APC7-dependent protein degradation pathways in neurons?

Investigating APC7-dependent protein degradation pathways in neurons requires specialized methodological approaches:

• Genetic models: Generate and utilize mouse models harboring patient-specific Anapc7 mutations. These models can be created by deleting specific exons (e.g., exons 4-6) to mimic human mutations .

• Biochemical activity assays: Isolate the APC from brain tissue via immunoprecipitation using antibodies against APC components (such as anti-APC3). Supply purified ubiquitin, Cdh1, E2 enzymes, E1 (Ube1), and fluorescent substrates to complete the ubiquitination reaction in vitro. Compare the ubiquitin ligase activity of APC isolated from control versus APC7 mutant brains .

• Proteomics approaches: Perform proteomics analyses of the brain from mice harboring APC7 mutations to identify APC7-dependent substrates. This approach successfully identified Ki-67 as an APC7-dependent substrate of the APC in post-mitotic neurons .

• Subcellular localization studies: Investigate the subcellular distribution of APC7 in fractionated brain lysates to determine its localization. Studies have shown that APC7 and APC3 are enriched in the nucleus relative to other APC subunits in the mouse cerebral cortex and cerebellum .

• Developmental activity assays: Compare APC activity between embryonic and adult brain tissues to understand how APC7 function changes during development. For example, immunoprecipitated APC from embryonic brain shows higher activity than APC isolated from adult mouse brain in ubiquitination assays .

What is the relationship between APC7 expression and breast cancer progression?

The relationship between APC7 expression and breast cancer progression reveals important correlations with several clinicopathological parameters:

• Inverse relationship with malignant characteristics: Loss of APC7 expression is more common in breast carcinoma cases with poor prognostic parameters or malignant characteristics. Negative APC7 expression is more frequent when histologic grade, nuclear grade, or mitotic number is elevated .

• Association with proliferation markers: The frequency of APC7 negative expression is higher in high Ki-67 groups than in low Ki-67 groups. Since Ki-67 is a well-established marker of cellular proliferation, this suggests a relationship between APC7 loss and increased proliferative activity in breast cancer .

• Correlation with aneuploidy: APC7 negative expression is more common in aneuploid groups than in diploid groups, suggesting a potential role for APC7 in maintaining chromosomal stability .

• Hypothesized mechanism: These findings suggest that dysregulation of APC activity, possibly through downregulation of APC7, may be associated with tumorigenesis in breast cancer. Given the role of the APC in cell cycle regulation and protein degradation, reduced APC7 expression might contribute to aberrant cell division and genomic instability .

How can researchers quantitatively assess APC7 expression in tumor samples?

Quantitative assessment of APC7 expression in tumor samples requires standardized scoring methods:

• Immunohistochemical scoring system: Implement a combined intensity and proportion scoring system. Intensity scores typically range from 0 (no staining) to 3+ (strong staining), while proportion scores represent the estimated percentage of stained cells (e.g., 0 = 0%, 1+ = 0-25%, 2+ = 25-50%, and 3+ = >50%) .

• Defining expression status: Establish clear criteria for what constitutes positive versus negative expression. For example, a summed intensity and proportion score of ≥3+ might be defined as positive APC7 expression, whereas a score of ≤2+ could be defined as negative .

• Reference controls: Include unstained tissue samples as negative controls and strongly stained tissue samples as positive controls in each staining procedure to ensure consistent scoring across different batches of samples .

• Cell counting methodology: For correlation with proliferation markers like Ki-67, define a labeling index as the percentage of positively stained cells in multiple high-power fields (×400), counting at least 1000 cells per field .

• Comparative analysis: Compare APC7 expression with other established prognostic markers (such as ER status) to contextualize findings within known prognostic frameworks .

How does APC7 contribute to the structural and functional integrity of the APC complex?

APC7's contribution to the structural and functional integrity of the APC complex involves several sophisticated mechanisms:

What are the challenges in developing highly specific APC7 antibodies for research?

Developing highly specific APC7 antibodies presents several technical challenges:

• Epitope selection: The APC7 protein contains multiple TPR motifs that share structural similarities with other proteins, making epitope selection challenging. Choosing unique regions like the carboxy terminus can improve specificity .

• Cross-reactivity with related proteins: The APC complex contains multiple subunits with similar structural domains, increasing the risk of antibody cross-reactivity. Rigorous validation is required to ensure specificity to APC7 rather than other APC components .

• Species conservation issues: APC7 is highly conserved across species (e.g., mouse APC7 has 97.7% homology with its human counterpart), which can make developing species-specific antibodies difficult. This high conservation can be advantageous for cross-species studies but problematic when species specificity is required .

• Validating functional relevance: Beyond simple binding, antibodies must be validated for their ability to recognize functionally relevant forms of APC7 within the intact APC complex. Immunoprecipitation experiments should demonstrate that APC7 antibodies can precipitate associated complex components like APC3 and APC6 .

• Application-specific optimization: Antibodies performing well in one application (e.g., Western blotting) may not work optimally in others (e.g., immunohistochemistry or immunoprecipitation), necessitating extensive application-specific optimization .

How might APC7 antibodies be utilized to explore developmental neurological disorders?

APC7 antibodies offer promising applications for investigating developmental neurological disorders:

• Diagnostic biomarker development: The connection between APC7 loss and intellectual disability syndromes suggests that APC7 antibodies could be used to develop diagnostic tools for certain neurodevelopmental disorders, particularly in populations where genetic testing has identified mutations in the ANAPC7 gene .

• Mechanistic studies: APC7 antibodies can help elucidate the mechanistic consequences of APC7 mutations by examining how these mutations affect:

  • Protein-protein interactions within the APC complex

  • Subcellular localization patterns in neuronal cells

  • Accumulation of APC substrates like Ki-67 in heterochromatin regions

• Tracking developmental expression: Using APC7 antibodies to track expression patterns during critical windows of brain development could reveal timing-specific requirements for APC7 function. This approach could identify when and where APC7 deficiency first manifests cellular abnormalities .

• Therapeutic target identification: Investigating APC7-dependent substrates may reveal potential therapeutic targets for neurodevelopmental disorders associated with APC7 dysfunction. Proteins that abnormally accumulate due to APC7 deficiency could represent novel intervention points .

What is the current understanding of APC7's role in chromatin regulation and how can it be further investigated?

The emerging role of APC7 in chromatin regulation presents exciting research opportunities:

• Current understanding: APC7 is required for the function of Cdh1-APC in neurons to regulate the chromatin-associated protein Ki-67. Upon APC7 loss, deregulated neuronal Ki-67 localizes predominantly to constitutive heterochromatin, suggesting APC7 plays a critical role in heterochromatin regulation during brain development .

• Chromatin immunoprecipitation sequencing (ChIP-seq): Researchers can use APC7 antibodies for ChIP-seq experiments to identify genomic regions where APC7 or its substrates associate with chromatin. This could reveal specific genes or regulatory elements affected by APC7 dysfunction .

• Proximity labeling approaches: Techniques like BioID or APEX2 proximity labeling, combined with APC7 antibodies for validation, can identify proteins in close proximity to APC7 within chromatin-associated complexes, potentially revealing novel interaction partners .

• Single-cell approaches: Combining APC7 antibody staining with single-cell transcriptomics or chromatin accessibility assays could reveal cell type-specific roles of APC7 in chromatin regulation during development .

• Functional rescue experiments: Testing whether wild-type APC7 can rescue chromatin abnormalities in cells expressing mutant APC7 could help establish causal relationships between APC7 function and specific chromatin regulatory mechanisms .