cyb561a3a Antibody

What is CYB561A3 and what is its primary function in cellular biology?

CYB561A3 functions as a transmembrane reductase that utilizes ascorbate as an electron donor in the cytoplasm. It transfers electrons across membranes to reduce iron cations Fe(3+) into Fe(2+) in the lumen of late endosomes and lysosomes. The reduced iron can then be extruded from these compartments to the cytoplasm through divalent metal-specific transporters. This process makes CYB561A3 a crucial player in endosomal and lysosomal cellular iron homeostasis .

What are the alternative names for CYB561A3 in scientific literature?

The protein is known by several alternative designations in research papers and databases, including:

• CYBASC3

• PSEC0259

• Lysosomal membrane ascorbate-dependent ferrireductase CYB561A3

• Cytochrome b ascorbate-dependent protein 3

• Cytochrome b561 family member A3

• Lysosomal cytochrome b

• LCytb

What types of CYB561A3 antibodies are available for research applications?

Currently, several types of antibodies targeting CYB561A3 are available for research purposes. One prominently described example is a rabbit polyclonal antibody suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) and Western blot (WB) applications. This particular antibody was generated using a recombinant fragment protein within Human CYB561A3 (amino acids 200 to C-terminus) as the immunogen .

How does CYB561A3 expression correlate with pathological conditions based on current research?

While studies specifically on CYB561A3 are still emerging, research on the CYB561 family indicates significant roles in cancer pathology. Elevated CYB561 expression has been associated with poor prognosis in breast cancer patients, particularly in HER2-positive subtypes. CYB561 appears to influence cancer progression through multiple mechanisms, including macrophage M2 polarization in the breast cancer microenvironment . Additionally, CYB561 shows involvement in neuroendocrine differentiation in castration-resistant prostate cancer, suggesting diverse roles across different cancer types .

What is known about the molecular interaction network of CYB561A3?

Proteomic studies have revealed that CYB561 interacts with H2AFY (macroH2A1), a histone H2A variant gene involved in metabolic functions, transcriptional gene regulation, and DNA damage response. This interaction has functional significance, as CYB561 regulates H2AFY protein expression by inhibiting its ubiquitination. Subsequently, this regulation influences downstream pathways, particularly the expression of NF-κB in HER2-positive breast cancer . The specific interaction partners of CYB561A3 warrant further investigation to fully characterize its unique molecular network distinct from other family members.

What is the relationship between CYB561A3 and iron metabolism in pathological conditions?

CYB561A3 plays a critical role in iron homeostasis through its ferrireductase activity. In cancer cells, altered expression of CYB561 affects intracellular ferrous iron concentration. Research in neuroendocrine prostate cancer has shown that depletion of CYB561 reduces intracellular Fe(2+) levels, suggesting its importance in maintaining iron availability for cancer cell metabolism . The manipulation of CYB561A3 expression could potentially influence cancer progression through iron-dependent pathways, making it an interesting target for therapeutic development.

What are the optimal protocols for CYB561A3 detection in human tissue samples?

For immunohistochemistry on paraffin-embedded tissues (IHC-P), the following protocol has been validated:

• Section preparation: Cut paraffin-embedded samples into 4-μm-thick slices

• Processing: Bake, dewax, and dehydrate sections

• Peroxidase blocking: Treat with 3% H₂O₂ solution

• Antigen retrieval: Use 0.01 mol/L citrate buffer

• Blocking: Apply 3% goat serum

• Primary antibody: Add CYB561A3 antibody at 1:100 dilution and incubate at 4°C overnight

• Secondary antibody: Incubate at 37°C for 20 minutes

• Development: Use 3,3'-diaminobenzidine for color development

• Counterstain: Apply hematoxylin

• Mounting: Seal with neutral gum

For quantification, the Immunoreactive Score system can be used, where "score ≤ 3" is regarded as negative expression and "score > 3" is considered positive expression.

What are the validated approaches for manipulating CYB561A3 expression in experimental models?

RNA interference has been successfully used to modulate CYB561 expression in research settings. The following approach has been documented:

• Design recombinant lentiviruses encoding short hairpin RNAs (shRNAs) specific to CYB561

• Example target sequences that have been effective include:

  • 5'-CGCCCACAGCACATCTTCTTT-3' (shRNA#1)

  • 5'-GCACATCTTTGCGCTCGTCAT-3' (shRNA#2)

  • 5'-CCTGCTGGTTTACCGTGTCTT-3' (shRNA#3)

• Use scrambled-shRNA (e.g., 5'-TTCTCCGAACGTGTCACGTTT-3') as a negative control

• Transduce cells following manufacturer's protocols

• Verify knockout efficiency using quantitative real-time PCR (qRT-PCR) and western blotting

This methodology provides a reliable approach to study the functional consequences of CYB561A3 downregulation in cellular models.

What are the recommended techniques for quantifying CYB561A3 expression at protein and mRNA levels?

For comprehensive analysis of CYB561A3 expression, a combination of techniques is recommended:

Protein Level Quantification:

• Western Blotting Protocol:

  • Cell lysis: Use buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton x-100, and protease inhibitors

  • Protein determination: Bicinchoninic acid protein assay kit

  • Primary antibody: Anti-CYB561A3 at 1:1000 dilution

  • Loading control: α-tubulin (1:10,000)

  • Detection: Enhanced chemiluminescence

mRNA Level Quantification:

• Quantitative Real-Time PCR (qRT-PCR):

  • RNA extraction: TRIzol reagent

  • Recommended primers for CYB561A3:

    • Forward: 5'-TATAGCGCATTTGAGCCCGAG-3'

    • Reverse: 5'-CCGGGTCAAGATGTAGAGCAC-3'

  • Internal reference (GAPDH):

    • Forward: 5'-TGACTTCAACAGCGACACCCA-3'

    • Reverse: 5'-CACCCTGTTGCTGTAGCCAAA-3'

Bioinformatic Analysis:
Researchers can also utilize databases such as TCGA, GEO, TNMplot, and Kaplan-Meier plotter to analyze CYB561A3 expression patterns across different tissues and disease states .

How is CYB561A3 antibody being used in breast cancer research?

The CYB561A3 antibody has become an important tool in breast cancer research, particularly in characterizing molecular subtypes and understanding disease progression mechanisms. Recent studies have employed immunohistochemistry with CYB561A3 antibodies to evaluate expression patterns across different breast cancer subtypes, revealing particularly elevated expression in HER2-positive breast cancer compared to other molecular subtypes .

Research applications include:

• Prognostic biomarker evaluation

• Molecular subtyping refinement

• Mechanism studies on iron metabolism in tumor microenvironments

• Therapeutic target exploration

What role does CYB561A3 play in the immune microenvironment of tumors?

CYB561 has demonstrated a significant association with macrophage M2 polarization in the breast cancer immune microenvironment. This finding has important implications for understanding tumor-immune interactions, as M2 macrophages typically promote tumor progression through immunosuppressive and pro-angiogenic activities.

The relationship between CYB561 and the immune microenvironment has been investigated through:

• CIBERSORT algorithm analysis to evaluate immune cell infiltration

• Single-cell RNA sequencing analysis

• Immunohistochemistry of serial sections to validate computational findings

These approaches have revealed that CYB561 expression correlates with macrophage polarization states, suggesting that it may influence tumor progression partly through modulation of the immune microenvironment .

What experimental models are suitable for studying CYB561A3 function in cancer?

Based on current research, the following experimental models have proven valuable for investigating CYB561 function in cancer:

In Vitro Models:

• HER2-positive breast cancer cell lines (BT474, SKBR-3)

• Triple-negative breast cancer cell lines (MDA-MB-231)

• Prostate cancer cell lines (LNCaP, PC-3)

In Vivo Models:

• Subcutaneous xenograft models in nude mice using:

  • SKBR-3 cells with CYB561 knockdown

  • PC-3 cells with CYB561 manipulation

Patient-Derived Models:

• Tissue microarrays of breast cancer samples for correlation of CYB561 expression with clinical outcomes

• Patient-derived xenografts for therapeutic studies

The selection of an appropriate model should be guided by the specific research question, with consideration for the molecular subtype being investigated and the particular aspect of CYB561A3 function under study .

What are the emerging therapeutic possibilities targeting CYB561A3 in cancer?

Given the association of CYB561 with poor prognosis in certain cancer types, several therapeutic approaches warrant investigation:

• Small molecule inhibitors targeting the ferrireductase activity of CYB561A3

• Antibody-drug conjugates using anti-CYB561A3 antibodies for targeted delivery

• Iron chelation therapy combined with CYB561A3 inhibition

• RNA interference-based therapeutics to downregulate CYB561A3 expression

• Targeting the CYB561A3-H2AFY axis in HER2-positive breast cancer

Preliminary research suggests that inhibition of CYB561 can reduce cancer cell proliferation, migration, and invasion in vitro, as well as suppress tumor growth in vivo, positioning it as a promising therapeutic target .

What methodological advances would enhance CYB561A3 research?

Several technological and methodological advancements could significantly advance CYB561A3 research:

• Development of highly specific monoclonal antibodies against different epitopes of CYB561A3

• CRISPR-Cas9 gene editing techniques for precise manipulation of CYB561A3 in various cell models

• Advanced imaging techniques to visualize CYB561A3 localization and dynamics in real-time

• Improved mass spectrometry methods for comprehensive analysis of CYB561A3 interaction partners

• Single-cell technologies to characterize CYB561A3 expression heterogeneity within tumors

• Development of conditional knockout mouse models specific to CYB561A3

These methodological advances would provide more precise tools for investigating the biological functions and disease associations of CYB561A3 .

How might iron metabolism and CYB561A3 function intersect with emerging cancer therapies?

The role of CYB561A3 in iron metabolism presents intriguing possibilities for combination therapies:

• Integration with immunotherapy: As CYB561 appears to influence macrophage polarization, combining CYB561A3 inhibitors with immune checkpoint inhibitors might enhance anti-tumor immune responses

• Ferroptosis induction: CYB561A3 inhibition could potentially sensitize cancer cells to ferroptosis-inducing agents by disrupting iron homeostasis

• Metabolism-targeted therapies: The intersection between iron metabolism and cancer cell metabolic dependencies suggests potential synergy between CYB561A3 inhibitors and metabolic pathway inhibitors

• Nanomedicine approaches: Iron oxide nanoparticles could be employed as both imaging agents and therapeutic carriers in conjunction with CYB561A3-targeted therapies

Research in these directions would benefit from integrated approaches combining molecular biology, immunology, and medicinal chemistry to develop effective therapeutic strategies targeting the CYB561A3-iron metabolism axis in cancer .