CYB561B Antibody

What is CYB561 and what are its primary biological functions?

CYB561 (Cytochrome b561) belongs to the cytochrome b561 family, member A1, located at chromosome 17q23.3. It functions primarily as an electron transfer protein that receives electrons from ascorbic acid and transfers them across membranes. The physiological functions facilitated by CYB561 include stress defense, cell wall modifications, iron metabolism, tumor suppression, and various neurological processes, including memory retention . CYB561 has been found to be highly expressed in breast cancer tissues and correlates with poor prognosis, while showing low expression in endometrial and ovarian cancers, also correlating with poor outcomes .

What expression patterns of CYB561 are observed in different cancer types?

CYB561 shows variable expression patterns across different cancer types. Analysis of cancer databases has revealed that breast cancer has the highest number of cases with CYB561 overexpression (n=12), followed by bladder cancer (n=2), with only single cases identified in other cancers including brain and CNS cancer, leukemia, lymphoma, ovarian cancer, and sarcoma . The mRNA expression of CYB561 in breast cancer is significantly higher than in normal tissues, as confirmed through the UALCAN database . Additionally, elevated CYB561 expression has been observed across different breast cancer stages, histological subtypes, and appears to be influenced by factors such as patient race and menopausal status .

How does CYB561 expression correlate with breast cancer molecular subtypes?

The expression of CYB561 varies significantly across different molecular subtypes of breast cancer. Immunohistochemical staining has demonstrated that HER2-positive breast cancer exhibits notably elevated CYB561 expression compared to other molecular subtypes . This finding is supported by transcript analysis of the TCGA database, which also showed the highest levels of CYB561 expression in HER2-positive breast cancer . The percentage of CYB561 positivity is higher in HER2-positive breast cancer as demonstrated by tissue microarray data . Importantly, high expression of CYB561 correlates with poorer prognosis specifically in HER2-positive breast cancer compared to other molecular subtypes .

What are the appropriate detection methods for CYB561?

Several methods are used to detect CYB561 protein and mRNA in research settings:

• Immunohistochemistry (IHC): For tissue samples, IHC can be performed using specific antibodies against CYB561 (e.g., rabbit anti-CYB561, 1:50 dilution, ThermoFisher, PA5-53228) with standard protocols including antigen retrieval with citrate buffer, blocking with goat serum, and visualization with diaminobenzidine solution .

• Western Blot: For protein detection in cell lysates, anti-CYB561 antibodies (e.g., Cat #PA5-53228, 1:1000, Thermo Fisher) can be used following standard protocols including SDS-PAGE separation, transfer to PVDF membranes, and detection with appropriate secondary antibodies .

• ELISA: Sandwich enzyme immunoassays are available for quantitative measurement of CYB561 in human serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids .

• RT-qPCR: For mRNA expression analysis, the 2^-ΔΔCt method (Livak method) is commonly used for relative quantification .

What is the mechanism by which CYB561 regulates H2AFY in cancer cells?

CYB561 has been shown to interact with H2AFY (also known as macroH2A1), a histone H2A variant gene involved in metabolic functions, transcriptional gene regulation, and DNA damage response . The interaction between CYB561 and H2AFY was initially identified through semi-quantitative mass spectrometry proteomic analysis of CYB561 immunoprecipitation products and subsequently confirmed by both exogenous and endogenous co-immunoprecipitation experiments .

Mechanistically, CYB561 regulates H2AFY expression by inhibiting its ubiquitination. When CYB561 is overexpressed, it extends the protein half-life of H2AFY, as demonstrated in experiments using the protein synthesis inhibitor cycloheximide (CHX) . Additionally, the proteasomal inhibitor MG132 restored H2AFY expression, suggesting that CYB561 stabilizes H2AFY through interference with the ubiquitin-proteasome system . Direct evidence showed that CYB561 overexpression decreased H2AFY ubiquitination in HEK293T cells . Furthermore, stronger nuclear signals of H2AFY were detected when exogenous CYB561 was expressed, while nuclear H2AFY signals significantly decreased in CYB561-knockdown cells .

How does the CYB561-H2AFY interaction affect downstream signaling pathways?

The CYB561-H2AFY interaction has significant effects on downstream signaling pathways, particularly the NF-κB pathway:

• Previous studies have shown that H2AFY facilitates cancer cell responses to the TNFα-NF-κB pathway .

• Gene Ontology (GO) biological process enrichment analysis demonstrated that genes interacting with CYB561 are enriched in NF-κB signaling .

• Compared to control cells, levels of NF-κB are increased in CYB561-overexpressing cells .

• Cell fractionation and Western blot analyses revealed that while cytoplasmic levels of NF-κB decrease upon CYB561 overexpression, nuclear levels significantly increase, suggesting enhanced nuclear translocation and activation of NF-κB .

This signaling cascade reveals a mechanism by which CYB561 promotes cancer cell proliferation through the CYB561-H2AFY-NF-κB axis, particularly in HER2-positive breast cancer .

What is the prognostic significance of CYB561 expression in different cancer types?

CYB561 expression has significant prognostic implications, particularly in breast cancer:

• Kaplan-Meier analysis indicates poorer prognosis in cancer samples with high CYB561 expression .

• The prognostic impact of CYB561 is most pronounced in HER2-positive breast cancer, where high expression correlates with shorter survival compared to other molecular subtypes .

• TIMER database analysis confirms that high CYB561 expression is associated with poorer prognosis specifically in HER2-positive breast cancer .

• Tissue microarray data also suggest an unfavorable prognosis for HER2-positive breast cancer patients with high CYB561 expression, although statistical significance may be limited by sample size in some studies .

These findings collectively suggest that CYB561 expression levels could serve as a potential prognostic biomarker, particularly for patients with HER2-positive breast cancer.

How should researchers optimize immunohistochemical detection of CYB561?

For optimal immunohistochemical detection of CYB561 in research settings, consider the following protocol based on published methods:

• Sample preparation: Bake human breast cancer tissue microarrays in a 65°C oven for 2 hours followed by rehydration with dewaxing in gradient decreasing concentrations of xylene and ethanol in sequence .

• Antigen retrieval: Extract the antigen with citrate buffer (ZSGB-BIO, ZLI-9065) for 90 seconds in a pressure cooker to expose the epitope to the antibody .

• Blocking steps: Inhibit endogenous peroxidase activity with 3% hydrogen peroxide, followed by incubation with goat serum at 37°C for 30 minutes .

• Primary antibody incubation: Incubate tissue microarrays with rabbit anti-CYB561 (1:50 dilution, ThermoFisher, PA5-53228) overnight at 4°C .

• Secondary antibody and visualization: After washing with phosphate-buffered saline (PBS), stain with an appropriate secondary antibody at 37°C for 1 hour, followed by staining with diaminobenzidine solution and counterstaining with hematoxylin .

• Scoring method: Calculate the final IHC score for CYB561 as the sum of the product of the staining intensity score and the percentage score to enable quantitative analysis .

What are the best practices for studying CYB561-protein interactions?

To effectively study CYB561-protein interactions in research contexts:

• Immunoprecipitation (IP) followed by mass spectrometry: This approach can identify potential CYB561-interacting proteins. Overexpress CYB561 in an appropriate cell line (e.g., HEK293T) with proper controls, perform IP, and analyze the products using semi-quantitative mass spectrometry .

• Filtering criteria: To minimize false positives, implement stringent filtering criteria. For example, exclude proteins identified with fewer than four unique peptides from analysis, as described in previous studies .

• Validation through co-IP: Confirm identified interactions using both exogenous and endogenous co-immunoprecipitation experiments. This two-pronged validation approach provides more robust evidence of protein interactions .

• Functional validation: For key interactions (like CYB561-H2AFY), perform functional assays such as ubiquitination assays, protein half-life assessments using cycloheximide chase experiments, and proteasome inhibition studies with MG132 to establish the functional consequences of the interaction .

• Subcellular localization studies: Use immunofluorescence microscopy to determine how interactions affect the subcellular distribution of the proteins of interest, as demonstrated for the nuclear localization of H2AFY in the presence or absence of CYB561 .

What approaches are recommended for quantitative measurement of CYB561 in biological samples?

For quantitative measurement of CYB561 in biological samples, researchers should consider:

• ELISA: Sandwich enzyme immunoassays specifically designed for CYB561 detection allow quantitative measurement in human serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids . These assays employ antibodies specific for human CYB561 pre-coated onto microplates, followed by detection antibodies and appropriate visualization systems .

• Western blotting with quantification: For protein-level expression analysis in cell or tissue lysates, western blotting with anti-CYB561 antibodies (e.g., Cat #PA5-53228, 1:1000, Thermo Fisher) coupled with appropriate loading controls and densitometric analysis using software like ImageJ provides semi-quantitative assessment .

• RT-qPCR: For mRNA expression analysis, real-time fluorescence quantitative PCR using the 2^-ΔΔCt method (Livak method) offers relative quantification of CYB561 transcript levels .

• Bioinformatic approaches: For large-scale analysis, databases such as Oncomine, GEPIA, and UALCAN can be utilized to determine differential expression of CYB561 across various sample types, while tools like xiantao can analyze mRNA expression and survival data from sources like TCGA .

What are the emerging research directions for CYB561 antibodies in cancer research?

Based on current research findings, several promising directions for CYB561 antibody applications in cancer research are emerging:

• Therapeutic target validation: As CYB561 has been identified as a potential therapeutic target for HER2-positive breast cancer, antibodies that can block its function could be developed and tested for anti-cancer activity in preclinical models .

• Diagnostic applications: Given the differential expression of CYB561 across breast cancer molecular subtypes, antibody-based detection methods might be further developed for diagnostic or prognostic applications .

• Mechanistic studies: CYB561 antibodies can be instrumental in further elucidating the mechanisms by which CYB561 promotes cancer progression, particularly through its interactions with H2AFY and effects on the NF-κB pathway .

• Combination therapy approaches: Research could explore how targeting CYB561 might enhance the efficacy of existing HER2-targeted therapies such as trastuzumab, pertuzumab, and tyrosine kinase inhibitors in breast cancer treatment .

• Expansion to other cancer types: While the current focus is on breast cancer, similar approaches could be applied to investigate the roles of CYB561 in other cancers where altered expression has been observed .