NFYA3 Antibody

What is NFYA and why is it important in cellular biology?

NFYA (Nuclear transcription factor Y subunit alpha) is a key subunit of the heterotrimeric transcription factor NF-Y, which recognizes the 5'-CCAAT-3' box motif found in the promoters of target genes. NFYA is considered the regulatory and limiting subunit of the NF-Y complex as it contains the DNA-binding domain at the C-terminus that enables sequence-specific interactions. Functionally, NFYA can act as both an activator and repressor depending on its interacting cofactors. It plays critical roles in various biological processes, including regulating the transcription of the core clock component BMAL1 and controlling metabolic pathways such as lipogenesis .

How do NFYA antibodies differ from NFAT3 antibodies?

These antibodies target completely different proteins with distinct functions:

While both are transcription factors, they function in different cellular pathways and should not be confused when designing experiments .

What are the known splice variants of NFYA and their functional differences?

Two primary splice variants of NFYA have been identified:

• NFYAv1 (long form): Contains all exons including exon 3, which encodes 29 amino acids in the Q-rich transactivation domain

• NFYAv2 (short form): Lacks the 29 amino acids encoded by exon 3 in the Q-rich transactivation domain

Research has shown significant functional differences between these variants. NFYAv1 upregulates transcription of essential lipogenic enzymes ACACA and FASN, enhancing malignant behavior in triple-negative breast cancer (TNBC). NFYAv1 expression correlates with mesenchymal markers like vimentin, while NFYAv2 correlates with epithelial markers like E-cadherin. The balance in expression between these variants correlates with prognosis in breast cancer, with NFYAv1 playing a more critical role in maintaining malignant behavior in TNBC cells .

What are the validated applications for NFYA antibodies in research?

NFYA antibodies have been validated for multiple experimental applications:

When selecting an NFYA antibody, researchers should verify which applications have been validated for their specific experimental needs. Some antibodies may perform better in certain applications than others .

How can I design experiments to distinguish between NFYAv1 and NFYAv2 functions?

To investigate the functional differences between NFYAv1 and NFYAv2:

• Selective knockout/knockdown: Generate variant-specific deficient cells using CRISPR/Cas9 (targeting exon 3 for NFYAv1-specific deletion)

• Rescue experiments: In NFYA-deficient cells, reintroduce either NFYAv1 or NFYAv2 to identify variant-specific function restoration

• Dominant-negative mutant approach: Overexpress dominant-negative mutants of each variant to suppress their specific functions

• EMT induction experiments: Use factors like SNAIL overexpression or forskolin treatment to observe NFYA variant switching during epithelial-mesenchymal transition

• Lipid metabolism assays: Measure lipogenic enzyme expression and activity in cells expressing different NFYA variants

Recent research showed that NFYAv1-deficient cells exhibited suppressed growth similar to NFYA-deficient cells, while NFYAv2 overexpression failed to rescue the phenotype, indicating functional specialization .

How does NFYA regulate lipid metabolism and what are the implications for cancer research?

NFYA, particularly the NFYAv1 variant, is a critical regulator of lipid metabolism through direct transcriptional control of key lipogenic enzymes:

• Transcriptional regulation: NFYA binds to CCAAT boxes in the promoter regions of lipogenic genes including ACLY, ACACA, ACACB, and FASN

• Metabolic pathway control: NFYAv1 promotes de novo lipogenesis rather than fatty acid oxidation

• Cancer metabolism connection: In TNBC, NFYAv1 upregulates ACACA and FASN expression, leading to increased lipid synthesis necessary for cancer cell proliferation

Experimental evidence shows that NFYA deficiency reduces expression of lipogenic enzymes, and this reduction can be partially rescued by adding lipids to the culture medium. FASN inhibition via cerulenin completely inhibits TNBC cell growth and sphere-forming ability, similar to NFYA deficiency. This provides strong mechanistic evidence that the NFYAv1-lipogenesis axis is essential for TNBC malignant behavior and represents a potential therapeutic target .

What role does NFYA play in cardiomyocyte development and metabolism?

NFYA plays a crucial role in cardiomyocyte (CM) development and metabolism:

• Developmental necessity: CM-specific knockout of NFYa in mice causes pre-birth lethality with underdeveloped hearts, presenting noncompaction and hypoplastic phenotypes

• Cell composition regulation: NFYa deletion alters CM composition in the fetal heart by decreasing immature CMs and increasing trabecular and mature CMs

• Metabolic control: NFYa, together with cofactor SP2, directly controls expression of metabolic genes in CMs

• Proliferation effects: NFYa deletion leads to decreased CM proliferation, with a statistically significant reduction in pH3-positive CMs (3.8% vs 4.5% in controls)

• Redox balance: NFYa knockout CMs show increased reactive oxygen species (ROS) and disturbed glutathione metabolism

These findings demonstrate that NFYa functions as a critical regulator coordinating metabolism and proliferation during heart development, highlighting its importance in fetal heart growth and establishment of neonatal regenerative cardiomyocytes .

What are the optimal conditions for using NFYA antibodies in immunofluorescence experiments?

For optimal immunofluorescence staining with NFYA antibodies:

• Fixation: Use 4% paraformaldehyde (10-15 minutes at room temperature) to preserve nuclear structures

• Permeabilization: Apply 0.1-0.5% Triton X-100 for adequate nuclear penetration

• Blocking: Use 5% normal serum (matching secondary antibody host) with 1% BSA to reduce background

• Primary antibody: Dilute NFYA antibody 1:100-1:500 in blocking buffer; incubate overnight at 4°C

• Secondary antibody selection: Use high-sensitivity fluorophore-conjugated antibodies with minimal cross-reactivity

• Nuclear counterstain: Include DAPI or Hoechst to visualize nuclei, as NFYA is primarily nuclear

• Microscopy: Use confocal microscopy for optimal resolution of nuclear staining patterns

For validation, include appropriate positive controls (cells known to express NFYA) and negative controls (NFYA-deficient cells or primary antibody omission) .

How should I design ChIP experiments to investigate NFYA binding to lipogenic enzyme promoters?

For effective ChIP experiments studying NFYA binding to lipogenic enzyme promoters:

• Antibody selection: Use ChIP-seq grade NFYA antibodies validated for immunoprecipitation (IP)

• Cross-linking: Optimize formaldehyde cross-linking time (typically 10 minutes) for transcription factors

• Sonication conditions: Adjust to yield DNA fragments of 200-500 bp for optimal resolution

• IP conditions: Use stringent washing conditions to eliminate non-specific binding

• Primer design: Design primers flanking predicted CCAAT boxes in ACACA and FASN promoters

• Controls:

  • Input DNA (non-immunoprecipitated)

  • IgG negative control

  • Positive control (known NFYA target gene)

• Data analysis: Normalize to input and IgG control; compare to known NFYA binding sites

Recent research successfully employed CUT&RUN assay (a more sensitive alternative to ChIP) with IP-validated NFYA antibodies to demonstrate direct binding to ACACA and FASN promoter regions .

Why might I observe differences in NFYA antibody performance across different cell types?

Several factors can contribute to variable NFYA antibody performance across cell types:

• Expression level differences: NFYA expression varies significantly between cell types; for example, breast cancer cells show higher expression than normal mammary epithelial cells

• Splice variant prevalence: Different cell types preferentially express different NFYA variants:

  • Luminal and HER2-positive breast cancer cells predominantly express NFYAv2

  • Basal-like breast cancer cells express both variants

  • Mesenchymal cells express more NFYAv1

• Epitope accessibility: Nuclear density and chromatin state may affect antibody access to NFYA

• Post-translational modifications: Cell-type specific modifications may alter antibody recognition

• Cofactor interactions: NFYA interactions with different binding partners can mask antibody epitopes

To address these issues, validate antibodies in your specific cell type, consider using multiple antibodies targeting different NFYA epitopes, and include appropriate positive and negative controls .

What are common pitfalls when interpreting Western blot results for NFYA and how can they be avoided?

The predicted band size for NFYA is approximately 37 kDa, but observed sizes may vary due to post-translational modifications or variant-specific differences. Western blots have successfully detected NFYA in various cell lines including NIH/3T3, HeLa, 293T, and Jurkat cells at antibody dilutions of 1/1000 .

What is the significance of NFYA in cancer progression and potential therapeutic targeting?

Recent research has revealed NFYA as a potential therapeutic target, particularly in triple-negative breast cancer:

• Cancer-specific expression patterns: NFYA shows increased expression in breast cancer cells compared to normal mammary epithelial cells

• Variant-specific oncogenic roles: NFYAv1 (long form) specifically promotes malignant behavior in TNBC through lipogenesis regulation

• Mechanistic insights: NFYAv1 directly regulates transcription of essential lipogenic enzymes ACACA and FASN

• Therapeutic potential: Several advantages of targeting NFYAv1:

  • NFYAv1-deficient mice show no apparent developmental abnormalities, unlike lipogenic enzyme knockouts that cause embryonic lethality

  • NFYAv1 deficiency strongly suppresses malignant behavior in vitro and in vivo

  • Targeting NFYAv1 affects upstream lipogenic regulation, potentially avoiding resistance mechanisms

The research suggests NFYAv1 may be a safer therapeutic target than directly inhibiting lipogenic enzymes, as knockout of ACLY, ACACA, or FASN causes embryonic lethality, while Nfyav1-deficient mice develop normally .

How are new antibody technologies advancing NFYA research?

Recent technological advances are enhancing NFYA antibody applications:

• Recombinant antibody production: Rabbit recombinant monoclonal antibodies (like EPR9061) offer improved consistency and specificity for NFYA detection across multiple applications

• Deep screening approaches: New methods leverage platforms like Illumina HiSeq to screen ~10^8 antibody-antigen interactions within 3 days, accelerating discovery of high-affinity antibodies

• AlphaFold3 integration: Computational tools are improving antibody design by predicting structural interactions, though current success rates for antibody docking remain at 8.9-13.4%

• Single-cell applications: Advanced antibodies compatible with single-nucleus RNA-seq and ATAC-seq are enabling integrated analysis of NFYA's role in specific cell populations

• Spatial transcriptomics integration: Combining NFYA antibody staining with spatial transcriptomics has revealed cell-type specific functions, as demonstrated in cardiomyocyte development studies

These technologies are driving more precise characterization of NFYA variant functions and creating opportunities for targeted therapeutic development .

What are the emerging research questions about NFYA function that remain to be addressed?

Several important questions about NFYA function remain unexplored:

• Variant-specific interactomes: How do the protein interaction networks differ between NFYAv1 and NFYAv2, and how do these differences contribute to their distinct functions?

• Regulatory mechanisms: What controls the alternative splicing of NFYA, particularly during epithelial-mesenchymal transition?

• Tissue-specific roles: How does NFYA function vary across different tissues and developmental stages?

• Therapeutic targeting: What approaches can specifically target NFYAv1 without affecting NFYAv2 function?

• Metabolic integration: How does NFYA coordinate lipid metabolism with other metabolic pathways in normal and disease states?

• Epigenetic regulation: How does NFYA interact with chromatin modifiers to influence gene expression patterns?

Addressing these questions will require integrated approaches combining genomics, proteomics, metabolomics, and functional studies using the latest antibody technologies and genetic tools .

What are the validated cell lines and tissue types for NFYA antibody applications?

This data compilation is based on published research findings and product information sheets for commercially available NFYA antibodies .

What are the recommended protocols for different NFYA antibody applications?