CENPU Antibody

What is CENPU and what cellular functions does it perform?

CENPU, also known as MLF1IP, PBIP1, KLIP1, or CENP-50, is a 47.5 kDa protein (418 amino acid residues in humans) exclusively expressed in centromeres throughout the cell cycle . It is a component of the CENPA-NAC (nucleosome-associated) complex, which plays a central role in:

• Assembly of kinetochore proteins

• Mitotic progression

• Chromosome segregation

CENPU is notably expressed at high levels in the testis, fetal liver, thymus, bone marrow and at lower levels in the lymph nodes, placenta, colon and spleen . It serves as a substrate for polo-like kinase 1 (Plk1), and its phosphorylation induces ubiquitination and degradation before the metaphase/anaphase transition .

What are the standard applications for anti-CENPU antibodies?

Anti-CENPU antibodies have been validated and utilized in multiple applications:

The choice of application should be based on experimental objectives and the specific validation data available for the particular antibody .

How should researchers validate anti-CENPU antibodies for their specific research applications?

Proper validation of anti-CENPU antibodies is crucial for reliable results. A comprehensive validation approach includes:

• Specificity verification:

  • Western blot analysis to confirm band size (47.5 kDa for canonical CENPU)

  • Positive and negative control tissues/cell lines

  • Knockdown or knockout validation using shRNA or CRISPR-Cas9

• Application-specific validation:

  • For IHC: Test on formalin-fixed, paraffin-embedded tissues with appropriate antigen retrieval (EGTA buffer, pH 9.0 has been successfully used)

  • For WB: Optimize protein extraction methods (SDS lysis buffer is commonly effective)

• Cross-reactivity assessment:

  • Test on tissues/cells from target and non-target species

  • Examine potential cross-reactivity with related centromere proteins

For knockdown validation, lentiviral vectors carrying CENPU shRNA have been successfully used in multiple studies, with transfection efficiency monitored via GFP fluorescence .

What are the optimal conditions for using anti-CENPU antibodies in immunohistochemistry?

Successful IHC staining for CENPU requires careful optimization:

• Sample preparation:

  • 4-μm sections from formalin-fixed, paraffin-embedded tissues

  • Deparaffinization in xylene

  • Rehydration in descending ethylalcohol series (100%, 95%, 85%, 75%)

• Antigen retrieval:

  • Boiling in EGTA buffer (pH 9.0) for 25 min

  • Alternative: citric acid buffer (specific conditions may need optimization)

• Antibody concentration and incubation:

  • Primary antibody dilution: 1:50 has shown good results with commercial antibodies (e.g., Abcam ab117078)

  • Overnight incubation at 4°C for primary antibody

  • Secondary antibody systems like EnVision-HRP at 37°C for 20 min

• Visualization and scoring:

  • 3,3′-diaminobenzidine (DAB) staining with hematoxylin counterstaining

  • Semi-quantitative scoring using intensity scale (0-3) and percentage of positive cells

How can CENPU antibodies be employed in studying cancer progression mechanisms?

Multiple studies have implicated CENPU in cancer progression, making anti-CENPU antibodies valuable tools for oncology research:

• Expression correlation studies:

  • CENPU is overexpressed in multiple cancers including breast cancer, hepatocellular carcinoma (HCC), and nasopharyngeal carcinoma (NPC)

  • Anti-CENPU antibodies in IHC can stratify patients based on expression levels for correlation with clinical parameters and survival outcomes

• Functional mechanistic studies:

  • Co-immunoprecipitation using anti-CENPU antibodies has revealed interactions with important regulatory proteins like DUSP6

  • Antibodies can help elucidate signaling pathways affected by CENPU, including Notch and MAPK pathways

• Cell phenotype studies:

  • After CENPU knockdown or overexpression, antibodies can be used to:

    • Verify protein level changes

    • Detect downstream effects on proteins in related pathways

    • Monitor subcellular localization changes during cell cycle progression

In HCC research, CENPU has been shown to promote malignant biological processes through the Notch signaling pathway, while in NPC, CENPU negatively regulates DUSP6 expression to promote cancer development .

What considerations are important when using anti-CENPU antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) with anti-CENPU antibodies requires careful planning:

• Antibody selection:

  • Choose antibodies raised against different epitopes than those of suspected interacting proteins

  • Ensure the antibody has been validated for IP/co-IP applications

  • Rabbit polyclonal antibodies have shown success in previous CENPU co-IP studies

• Protocol optimization:

  • Cell lysis conditions should preserve protein-protein interactions

  • Incubate cell lysates with anti-CENPU antibodies at 4°C overnight

  • Use magnetic beads (rather than agarose) for cleaner results

  • Include appropriate washing steps to minimize non-specific binding

• Controls:

  • IgG negative control is essential to distinguish specific from non-specific binding

  • Input samples should be run alongside IP samples

  • Reciprocal co-IP (using antibodies against suspected interacting partners) strengthens interaction evidence

The interaction between CENPU and DUSP6 in nasopharyngeal carcinoma was confirmed using this approach, with both forward and reverse co-IP demonstrating the interaction .

How do anti-CENPU antibodies compare with antibodies against other centromere proteins?

The centromere complex contains multiple proteins, and understanding the differences between anti-CENPU and other anti-centromere antibodies is important:

• Specificity considerations:

  • CENPU antibodies target a specific centromere protein, unlike autoantibodies from patients which often recognize multiple centromere proteins

  • Anti-centromere autoantibodies (ACA) commonly target CENP-A, CENP-B, and CENP-C proteins in autoimmune conditions

  • When studying centromere biology broadly, complementary antibodies against multiple centromere proteins may be necessary

• Clinical relevance comparison:

  • Anti-CENP-A and anti-CENP-B antibodies have established diagnostic value for systemic sclerosis (SSc)

  • CENPU antibodies are primarily research tools without established diagnostic value

  • The sensitivity and specificity of anti-CENP-A and anti-CENP-B for SSc are 93% and 96.5% respectively

• Epitope considerations:

  • Anti-CENPU antibodies target specific epitopes on CENPU

  • Autoantibodies against centromere proteins often recognize multiple epitopes simultaneously

A comprehensive study comparing autoantibodies against CENP-A and CENP-B showed that in systemic sclerosis, the autoantibody response targets both components with similar amplitude, whereas in other autoimmune diseases, the response is predominantly against one component .

What are common troubleshooting approaches for weak or non-specific signals with anti-CENPU antibodies?

When experiencing issues with anti-CENPU antibodies, systematic troubleshooting is recommended:

• Weak signal solutions:

  • Increase antibody concentration (commercial antibodies have been successfully used at 1:50 for IHC)

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

  • Optimize antigen retrieval method (EGTA buffer, pH 9.0 has shown good results)

  • Consider signal amplification systems

• Non-specific signal solutions:

  • Increase blocking duration and concentration

  • Use more stringent washing conditions

  • Titrate antibody to optimal concentration

  • Validate antibody specificity via knockdown experiments

  • For IHC, use appropriate negative controls (PBS instead of primary antibody)

• Tissue-specific considerations:

  • Different tissue fixation methods may affect epitope accessibility

  • Endogenous expression levels vary across tissues (highest in testis, fetal liver, thymus, and bone marrow)

  • Background may differ between cancer and normal tissues due to differential expression

How can researchers interpret contradictory CENPU expression data across different cancer studies?

Researchers may encounter discrepancies in CENPU expression or function across studies. These inconsistencies can be addressed by:

An integrated approach using multiple databases (TCGA, GEO, GEPIA) followed by experimental validation has been successfully employed to establish CENPU's role in hepatocellular carcinoma .

What are the emerging applications of anti-CENPU antibodies in studying cancer therapeutic targets?

Recent research indicates CENPU's potential as a therapeutic target, opening new applications for anti-CENPU antibodies:

• Target validation studies:

  • Anti-CENPU antibodies can verify protein knockdown efficiency in preclinical models

  • Expression levels before and after experimental treatments can be monitored

  • Changes in subcellular localization upon drug treatment can be tracked

• Mechanism of action studies:

  • CENPU has been implicated in the Notch signaling pathway in HCC

  • In NPC, CENPU interacts with and negatively regulates DUSP6

  • Antibodies can help elucidate these pathways and identify potential intervention points

• Biomarker development:

  • CENPU expression correlates with poor prognosis in multiple cancers

  • Anti-CENPU antibodies can help identify patient subgroups who might benefit from targeted therapies

  • Monitoring CENPU expression changes during treatment may provide insights into response mechanisms

• Combination therapy research:

  • CENPU knockdown affects multiple downstream pathways including p38/MAPK and ERK1/2

  • Anti-CENPU antibodies can help determine optimal combinations with existing targeted therapies

What special considerations apply when using anti-CENPU antibodies for gene expression profiling studies?

When incorporating anti-CENPU antibodies in multi-omics studies:

How do researchers reconcile differences between in vivo and in vitro findings when using CENPU antibodies?

Discrepancies between in vivo and in vitro CENPU studies are common and can be addressed by:

• System-specific optimization:

  • Antibody concentration and incubation conditions may differ between cell lines and tissue sections

  • For tissues, antigen retrieval methods are critical (EGTA buffer, pH 9.0 has been effective)

  • For cell lines, fixation methods affect epitope accessibility

• Context-dependent expression:

  • CENPU expression is cell cycle-dependent in normal cells

  • Cancer tissues may show dysregulated expression patterns

  • Microenvironment factors in vivo may affect expression and not be replicated in vitro

• Functional validation approaches:

  • Complement antibody-based detection with functional assays

  • Use gene knockdown/knockout in both systems

  • Consider xenograft models to bridge in vitro and in vivo findings

• Multidimensional analysis:

  • Combine antibody-based detection with other methodologies

  • For in vivo studies, correlate CENPU expression with multiple clinical parameters

  • For in vitro studies, examine effects on multiple cellular processes