CYB561C Antibody

What is CYB561 and why is it important in cancer research?

CYB561 (Cytochrome b561) is a transmembrane protein that has gained significant attention in cancer research, particularly in breast cancer studies. Recent investigations have demonstrated that CYB561 is highly expressed in breast cancer tissues compared to normal tissue samples, with elevated expression correlating with poor prognosis . The protein has been shown to promote cancer cell proliferation, migration, and invasion in vitro, while CYB561 knockdown inhibits these oncogenic properties .

The importance of CYB561 as a research target stems from its potential role as both a prognostic biomarker and therapeutic target, especially in HER2-positive breast cancer where it shows particularly high expression levels . Additionally, CYB561 has been associated with macrophage M2 polarization in the breast cancer microenvironment, suggesting its involvement in tumor-immune interactions that could influence cancer progression .

What detection methods are available for studying CYB561 expression in tissue samples?

Several complementary methods can be employed to detect and quantify CYB561 expression in research samples:

• Immunohistochemistry (IHC): This technique allows visualization of CYB561 protein expression in tissue sections and is particularly useful for comparing expression between cancer and normal tissues. Studies have successfully employed IHC to demonstrate elevated CYB561 expression in ductal and lobular carcinomas compared to normal breast tissue .

• ELISA: Quantitative sandwich enzyme immunoassay techniques are available for measuring CYB561 levels in human serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids. Commercial ELISA kits offer detection ranges of approximately 31.25-2000 pg/mL with sensitivity typically below 15.6 pg/mL .

• Western Blotting: For protein-level detection in cell or tissue lysates, antibodies specific to human CYB561 can be utilized in western blot applications. Commercially available polyclonal antibodies have been developed against specific amino acid regions (e.g., AA 72-121) of the CYB561 protein .

• RNA expression analysis: Transcriptome analysis using RT-qPCR or RNA-seq can be employed to measure CYB561 mRNA expression levels, as demonstrated in studies utilizing databases such as The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) .

How should I select the appropriate anti-CYB561 antibody for my experiments?

When selecting an anti-CYB561 antibody for research applications, consider the following key factors:

• Target epitope specificity: Choose antibodies that target specific amino acid sequences of CYB561, such as those targeting AA 72-121 regions, which have demonstrated high specificity in previous studies . This is particularly important for distinguishing between potential isoforms or related proteins.

• Host species compatibility: Consider the host species of the antibody (e.g., rabbit, mouse) in relation to your experimental design, especially for co-staining experiments where antibody combinations from different host species may be required .

• Application validation: Select antibodies that have been validated for your specific application (Western blotting, IHC, immunofluorescence, etc.). For example, certain polyclonal antibodies have been specifically validated for Western blotting applications against human, guinea pig, and monkey samples .

• Cross-reactivity profile: Review the predicted reactivity and percent identity information. Some antibodies show high identity (85-100%) across species including human, mouse, rat, dog, and others, which is valuable for comparative studies across model systems .

• Antibody format: Consider whether you need conjugated or unconjugated antibodies based on your detection method. For instance, unconjugated antibodies are typically used for Western blotting followed by secondary antibody detection .

What controls should I include when using CYB561 antibodies in my experiments?

Implementing proper controls is crucial for ensuring the validity and reliability of experiments utilizing CYB561 antibodies:

• Positive tissue controls: Include tissue samples known to express CYB561, such as breast cancer tissues (particularly HER2-positive subtypes) which have been shown to express high levels of CYB561 .

• Negative controls: Include samples where primary antibody is omitted but all other steps are performed identically to assess non-specific binding of secondary antibodies or detection reagents.

• Expression validation controls: When studying CYB561 knockdown or overexpression, validate the change in expression using both protein (Western blot) and mRNA (qPCR) methods to confirm the specificity of your antibody and the effectiveness of your intervention .

• Cross-reactivity controls: If working with non-human samples, use species-matched negative controls and validate antibody specificity using sequence alignment information, as some antibodies show cross-reactivity with mouse, rat, and other species at 85-92% sequence identity .

• Concentration gradient: Perform antibody titration experiments to determine optimal antibody concentration that maximizes specific signal while minimizing background, especially important for quantitative applications like ELISA .

How can I investigate the relationship between CYB561 expression and macrophage M2 polarization in the tumor microenvironment?

To investigate the relationship between CYB561 and macrophage M2 polarization in the tumor microenvironment, a multi-faceted approach combining computational and experimental methods is recommended:

• Single-cell RNA sequencing analysis: Apply single-cell sequencing to simultaneously evaluate CYB561 expression and macrophage polarization states within the tumor microenvironment. This approach has successfully been used to correlate CYB561 expression with specific immune cell infiltration patterns .

• CIBERSORT algorithm application: Utilize computational deconvolution methods like CIBERSORT to estimate the abundance of macrophage subtypes in bulk tumor samples and correlate these with CYB561 expression levels. This computational approach can be particularly useful for analyzing large public datasets .

• Serial section immunohistochemistry: Perform immunohistochemistry on serial tissue sections using CYB561 antibodies alongside M2 macrophage markers (e.g., CD163, CD206) to visualize spatial relationships between CYB561-expressing cells and M2-polarized macrophages in the tumor microenvironment .

• Co-culture experiments: Establish co-culture systems with cancer cells expressing varying levels of CYB561 (through knockdown or overexpression) and monocytes/macrophages to directly assess the influence of CYB561 on macrophage polarization. Measure M2 markers by flow cytometry or qPCR following co-culture .

• Conditioned media experiments: Collect conditioned media from CYB561-manipulated cancer cells and treat macrophages to determine if secreted factors regulated by CYB561 influence macrophage polarization, providing insight into potential paracrine signaling mechanisms.

What molecular mechanisms explain CYB561's role in breast cancer progression, particularly in HER2-positive subtypes?

Recent research has begun to elucidate several molecular mechanisms through which CYB561 contributes to breast cancer progression, with particular relevance to HER2-positive subtypes:

• H2AFY ubiquitination pathway: CYB561 has been demonstrated to upregulate macroH2A (H2AFY) expression in HER2-positive breast cancer cells specifically through inhibition of H2AFY ubiquitination. This post-translational regulation represents a key mechanism by which CYB561 influences downstream signaling pathways .

• NF-κB signaling modulation: CYB561 regulates H2AFY expression, which in turn influences the expression of NF-κB, a critical transcription factor involved in inflammation and cancer progression. This CYB561-H2AFY-NF-κB axis represents a significant pathway in HER2-positive breast cancer .

• Tropomyosin 1 expression regulation: CYB561 has been observed to modulate downstream tropomyosin 1 expression, potentially affecting cytoskeletal organization and cell motility, which could explain its influence on cancer cell migration and invasion capabilities .

• Immune microenvironment modulation: The association between CYB561 expression and macrophage M2 polarization suggests that CYB561 may influence tumor progression indirectly by shaping the immune microenvironment to favor tumor-promoting conditions .

To investigate these mechanisms further, researchers should consider employing protein-protein interaction studies, ubiquitination assays, chromatin immunoprecipitation experiments, and pathway inhibition approaches to dissect the specific molecular interactions involved.

How can I develop effective CYB561 knockdown or overexpression models to study its function in cancer cells?

Developing robust CYB561 manipulation models is essential for functional studies. Here are methodological approaches for creating effective experimental systems:

• siRNA/shRNA knockdown optimization:

  • Design multiple siRNA sequences targeting different regions of CYB561 mRNA

  • Validate knockdown efficiency at both mRNA (qPCR) and protein levels (Western blot using specific anti-CYB561 antibodies)

  • Establish dose-response relationships to determine optimal siRNA concentrations

  • For stable knockdown, clone validated shRNA sequences into lentiviral vectors with appropriate selection markers (puromycin, GFP, etc.)

• CRISPR-Cas9 gene editing:

  • Design guide RNAs targeting early exons of CYB561

  • Validate editing efficiency using T7 endonuclease assay or next-generation sequencing

  • Isolate and verify single-cell clones with confirmed mutations

  • Validate complete protein loss using specific antibodies in Western blot analysis

• Overexpression systems:

  • Clone the complete CYB561 coding sequence into mammalian expression vectors

  • Include epitope tags (FLAG, HA, etc.) for detection if necessary

  • Validate expression levels using qPCR and Western blotting

  • Consider inducible expression systems (e.g., Tet-On) for controlled overexpression studies

• Rescue experiments:

  • In knockdown models, reintroduce wild-type or mutant CYB561 to confirm specificity of observed phenotypes

  • Use silent mutations in overexpression constructs to make them resistant to siRNA when performing rescue experiments

• Model validation:

  • Confirm functional consequences of CYB561 manipulation by assessing established phenotypes like proliferation, migration, and invasion

  • Validate downstream molecular changes, particularly H2AFY levels and ubiquitination status in HER2-positive models

What techniques can I use to investigate CYB561's impact on protein ubiquitination, particularly for H2AFY in HER2-positive breast cancer?

To study CYB561's effect on protein ubiquitination, particularly its reported impact on H2AFY in HER2-positive breast cancer, the following methodological approaches are recommended:

• Ubiquitination assays:

  • Immunoprecipitate H2AFY from cells with varying CYB561 expression levels

  • Perform Western blotting with anti-ubiquitin antibodies to detect ubiquitinated H2AFY

  • Use proteasome inhibitors (MG132, bortezomib) to prevent degradation of ubiquitinated proteins during experiments

  • Compare ubiquitination patterns between control and CYB561-manipulated cells

• Cycloheximide chase assays:

  • Treat cells with cycloheximide to inhibit new protein synthesis

  • Collect samples at various time points to monitor H2AFY protein degradation rates

  • Compare protein half-life in cells with different CYB561 expression levels

  • Western blot analysis using anti-H2AFY antibodies can quantify protein stability differences

• Ubiquitin mutant overexpression:

  • Express mutant ubiquitin constructs (e.g., K48R, K63R) to identify specific ubiquitin linkage types involved

  • Co-express these with CYB561 and H2AFY to determine which ubiquitin modifications are affected by CYB561

• In vitro deubiquitination assays:

  • Purify ubiquitinated H2AFY from cells

  • Incubate with recombinant deubiquitinating enzymes in the presence or absence of purified CYB561

  • Analyze changes in ubiquitination patterns to determine if CYB561 directly affects deubiquitination activities

• Mass spectrometry analysis:

  • Perform immunoprecipitation of H2AFY followed by mass spectrometry

  • Identify ubiquitination sites and quantify changes in ubiquitination at specific lysine residues

  • Compare ubiquitination profiles between samples with different CYB561 expression levels

• Identification of E3 ligases:

  • Perform protein interaction studies to identify which E3 ligases interact with H2AFY

  • Assess whether CYB561 competes with or modifies these interactions

  • Use co-immunoprecipitation with specific antibodies against candidate E3 ligases and Western blotting to validate interactions

How can I leverage bioinformatic approaches to study CYB561's role across different cancer types and datasets?

Bioinformatic analyses provide powerful approaches to investigate CYB561's role across cancer types and identify patterns that may not be apparent in individual experiments:

• Multi-cancer expression analysis:

  • Utilize databases like UALCAN, The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) to compare CYB561 expression across multiple cancer types

  • Generate visualization of expression data using boxplots or heatmaps to identify cancer types with particularly high or low CYB561 expression

  • Statistical analysis should include appropriate multiple testing corrections when comparing across numerous cancer types

• Survival analysis stratification:

  • Implement Kaplan-Meier survival analysis using tools like Kaplan-Meier plotter

  • Stratify patients based on CYB561 expression levels (high vs. low)

  • Further stratify by cancer molecular subtypes (e.g., Luminal A, Luminal B, HER2-positive, triple-negative for breast cancer)

  • Calculate hazard ratios and p-values to quantify prognostic significance

• Gene co-expression network analysis:

  • Construct co-expression networks to identify genes whose expression patterns correlate with CYB561

  • Apply WGCNA (Weighted Gene Co-expression Network Analysis) to identify modules of co-expressed genes

  • Perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on co-expressed gene modules to identify biological processes associated with CYB561

• Immune infiltration correlation:

  • Use CIBERSORT, xCell, or similar algorithms to estimate immune cell composition in tumor samples

  • Correlate immune cell infiltration patterns with CYB561 expression levels

  • Focus particularly on macrophage populations (M1 vs. M2) given the reported association with M2 polarization

• Multi-omics integration:

  • Integrate CYB561 expression data with mutation, copy number variation, methylation, and proteomics data

  • Identify potential regulatory mechanisms affecting CYB561 expression

  • Use tools like cBioPortal to explore these multi-omics relationships

This comprehensive bioinformatic approach can provide insights into the broader biological context of CYB561 function and identify promising avenues for further experimental investigation across cancer types.