NFYA10 Antibody

What is NFYA10 and what are its primary functions in cellular processes?

NFYA10 (Nuclear Transcription Factor Y, alpha 10) is a highly conserved transcription factor that belongs to the NF-Y family. In Arabidopsis thaliana, NFYA10 functions in regulating lateral root development and gravitropic responses. It recognizes and binds to CCAAT motifs in promoters of target genes, thereby stimulating their transcription . NFYA10 directly regulates LAZY genes, which are linked to gravitropism, by targeting their promoter regions . The protein is predominantly expressed in the root vasculature, endodermis, and cortex cells, and is present during lateral root development from early stages through growth .

How does NFYA10 differ from other NF-YA family members?

NFYA10 belongs to clade D of the NF-YA transcription factor family. Phylogenetic analysis indicates that NFYA10 and NFYA2 arose from a relatively recent duplication specific to Brassicaceae . Unlike some other NF-YA proteins, NFYA10 is specifically regulated by the miRNA isoform miR169defg, which downregulates its expression . While many NF-YA proteins associate with tight dimers composed of NF-YB and NF-YC subunits to form trimers that bind DNA with high specificity, NFYA10 has a particular expression pattern in the root vasculature and plays a specific role in modulating the area explored by plant roots through its effects on lateral root development and gravitropism .

What detection methods are suitable for NFYA10 antibodies?

NFYA10 antibodies can be effectively used in multiple detection methods. Western blotting is a primary application, typically with dilutions around 1:1,000 . ELISA is another suitable method, with recommended dilutions of approximately 1:10,000 . For plant research, immunohistochemistry (IHC) and immunofluorescence (IF) can be employed to visualize NFYA10 expression patterns in specific cell types, such as the root vasculature, endodermis, and cortex cells . When conducting ChIP-qPCR experiments to identify direct targets of NFYA10, antibodies can be used to immunoprecipitate NFYA10-DNA complexes, focusing on TSS-proximal CCAAT boxes present in promoter regions of target genes .

How should I design experiments to study NFYA10's role in root development?

When designing experiments to investigate NFYA10's function in root development, consider the following methodological approach:

• Utilize transgenic plants expressing translational fusions such as pNF-YA10:GFP-NF-YA10 to visualize protein localization in root tissues

• Implement miRNA-resistant versions of NFYA10 (NF-YA10miRres) to study the effects of elevated NFYA10 expression without miR169 regulation

• Apply high-throughput phenotyping systems like ChronoRoot to analyze multiple root architecture parameters, including:

  • Main root (MR), lateral root (LR), and total root (TR) lengths

  • Number and density of lateral roots

  • Root growth speeds

  • Lateral root angles relative to gravitropic stimuli

• Perform ChIP-qPCR targeting CCAAT boxes in promoters of potential target genes, using gene bodies as negative controls to identify direct regulatory targets

• Include appropriate controls: wild-type plants, known mutants affecting root development (like lazy mutants), and plants with altered expression of other NF-YA family members

What controls should I include when validating NFYA10 antibody specificity?

To validate NFYA10 antibody specificity for research applications, implement the following controls:

• Positive tissue control: Use tissues with known NFYA10 expression, such as root vasculature, endodermis, and cortex cells in Arabidopsis

• Negative tissue control: Include tissues with minimal NFYA10 expression or use a knockout/knockdown model if available

• Pre-absorption control: Pre-incubate the antibody with excess purified NFYA10 antigen before immunostaining to demonstrate that staining is blocked when the antibody is neutralized

• Cross-reactivity assessment: Test the antibody against closely related NF-YA family members, particularly NFYA2, which is phylogenetically close to NFYA10

• Gene silencing validation: Compare antibody signals between wild-type samples and those with NFYA10 silenced through RNAi or CRISPR techniques

• Western blot verification: Confirm a single band of expected molecular weight before proceeding with more complex applications

How can I optimize Western blot conditions specifically for NFYA10 detection?

For optimal NFYA10 detection by Western blotting, follow these methodological guidelines:

• Sample preparation: Extract nuclear proteins using buffers containing phosphatase and protease inhibitors to preserve NFYA10 integrity

• Gel percentage: Use 10-12% SDS-PAGE gels to effectively resolve NFYA10 protein

• Transfer conditions: Employ wet transfer at 30V overnight at 4°C to ensure complete transfer of nuclear proteins

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation: Dilute NFYA10 antibody 1:1,000 in blocking solution and incubate overnight at 4°C

• Washing: Perform 4-5 washes with TBST, 5-10 minutes each

• Secondary antibody: Use anti-rabbit HRP-conjugated secondary antibody (as NFYA10 antibodies are typically rabbit-derived) at 1:5,000 dilution

• Detection method: Start with standard ECL detection; for low abundance samples, consider using more sensitive detection systems

• Exposure optimization: Begin with short exposures (30 seconds) and extend as needed to avoid overexposure

How can NFYA10 antibodies be utilized in ChIP experiments to identify target genes?

For effective ChIP experiments using NFYA10 antibodies, implement this methodological protocol:

• Crosslinking: Crosslink plant tissue with 1% formaldehyde for 10 minutes, followed by quenching with glycine

• Chromatin preparation: Isolate nuclei, sonicate chromatin to 200-500bp fragments, and confirm fragment size by gel electrophoresis

• Immunoprecipitation: Incubate sonicated chromatin with NFYA10 antibody overnight at 4°C (pre-clear chromatin with protein A/G beads before adding antibody)

• Washing and elution: Perform stringent washes to remove non-specific binding, then elute DNA-protein complexes

• Reversal of crosslinks: Incubate at 65°C overnight with proteinase K treatment

• DNA purification: Purify the immunoprecipitated DNA using column purification methods

• Target identification: Perform qPCR targeting specific CCAAT box-containing regions in promoters of interest, such as LAZY1 and LAZY2 genes

• Controls: Include input DNA (not immunoprecipitated), IgG control (non-specific antibody), and gene body regions (negative control) as shown in the research on LAZY gene regulation

• Data analysis: Calculate fold enrichment of promoter regions compared to gene body or IgG controls

• Validation: Confirm ChIP-qPCR results with functional studies such as gene expression analysis in NFYA10 overexpression or knockdown lines

What approaches help investigate interactions between NFYA10 and other transcription factors?

To investigate NFYA10 interactions with other transcription factors, employ these methodological strategies:

• Co-immunoprecipitation (Co-IP): Use NFYA10 antibodies to pull down protein complexes from nuclear extracts, followed by Western blotting with antibodies against suspected interacting partners

• Yeast two-hybrid assays: Create NFYA10 bait constructs to screen for interactions with other transcription factors, particularly NF-YB and NF-YC subunits which typically form heterotrimers with NF-YA proteins

• Bimolecular Fluorescence Complementation (BiFC): Generate fusion constructs of NFYA10 and potential interacting partners with split fluorescent protein fragments to visualize interactions in planta

• Proximity ligation assay: Detect protein-protein interactions in situ using NFYA10 antibodies and antibodies against potential interaction partners

• Sequential ChIP (Re-ChIP): Perform ChIP with NFYA10 antibody followed by a second immunoprecipitation with antibodies against suspected co-regulators to identify genomic regions bound by both factors

• Mass spectrometry analysis: Immunoprecipitate NFYA10 complexes using specific antibodies and identify interacting proteins through mass spectrometry

• DNA-protein interaction assays: Use electrophoretic mobility shift assays (EMSA) with purified NFYA10 protein to examine how interactions with other factors affect DNA binding specificity

How can I study the relationship between miRNA regulation and NFYA10 function?

To investigate miRNA regulation of NFYA10, implement the following experimental approaches:

• Expression analysis: Compare NFYA10 protein levels (using antibodies) and mRNA levels to identify post-transcriptional regulation

• miRNA-resistant constructs: Generate transgenic plants expressing NFYA10 lacking the 3'UTR region targeted by miR169 to create miRNA-resistant versions

• Root phenotyping: Analyze root architecture in wild-type versus miRNA-resistant NFYA10 plants using systems like ChronoRoot to quantify differences in:

  • Lateral root density

  • Root angle distribution

  • Root growth dynamics

• miRNA inhibition: Use target mimicry approaches to sequester miR169 and observe effects on NFYA10 protein levels using antibodies

• Reporter assays: Create reporter constructs containing the NFYA10 3'UTR fused to a reporter gene to monitor miRNA regulation

• Expression correlation: Analyze the inverse correlation between miR169 levels and NFYA10 protein expression across different tissues or conditions

• Functional analysis: Compare phenotypes of plants overexpressing miR169 with NFYA10 knockdown plants to confirm the specificity of the regulatory relationship

What factors might lead to inconsistent NFYA10 antibody detection results?

When encountering inconsistent results with NFYA10 antibodies, consider these potential factors and solutions:

• Protein degradation: Nuclear transcription factors can be sensitive to proteolysis. Use freshly prepared samples and include both protease and phosphatase inhibitors in extraction buffers

• Cross-reactivity: NFYA10 belongs to a family of similar proteins. Validate antibody specificity using knockout/knockdown controls and consider using antibodies targeting unique regions of NFYA10

• Post-translational modifications: NFYA proteins undergo modifications that may affect antibody recognition. Document experimental conditions carefully and consider using phospho-specific antibodies if relevant

• Expression levels: NFYA10 exhibits low expression levels in specific cell types . Concentrate samples appropriately and use sensitive detection methods

• Fixation artifacts: For immunohistochemistry, optimize fixation protocols as overfixation can mask epitopes

• Antibody batch variation: Validate new antibody lots against previous ones using positive control samples

• Isoform detection: Confirm which NFYA10 isoforms your antibody recognizes, as alternative splicing may occur

• miRNA regulation: NFYA10 is regulated by miR169 , which may cause expression variability across tissues or conditions. Document tissue source and growth conditions carefully

How can I differentiate between NFYA10 and other closely related NF-YA proteins?

To differentiate NFYA10 from other NF-YA family members in your experiments:

• Antibody selection: Choose antibodies raised against unique N-terminal or C-terminal regions of NFYA10 rather than the conserved DNA-binding domain

• Sequence analysis: Perform sequence alignments of NF-YA family members to identify unique epitopes for antibody selection

• Peptide competition assays: Pre-incubate antibodies with specific peptides from NFYA10 versus other NF-YA proteins to determine specificity

• Expression patterns: Utilize known differential expression patterns—NFYA10 shows specific expression in root vasculature, endodermis, and cortex cells

• Knockout validation: Test antibodies on tissues from nf-ya10 mutants compared to other nf-ya mutants

• Western blot resolution: Use high-resolution SDS-PAGE (gradient gels) to separate closely related NF-YA proteins based on slight molecular weight differences

• 2D gel electrophoresis: Separate NF-YA proteins by both isoelectric point and molecular weight for improved discrimination

• Mass spectrometry validation: Confirm antibody specificity by analyzing immunoprecipitated proteins with mass spectrometry to identify unique peptides

What methodological approaches help investigate NFYA10's role in plant gravitropism?

To investigate NFYA10's function in gravitropic responses, employ these methods:

• Gravitropic stimulation assays: Rotate plants 90° to horizontal position and measure root bending angles over time using time-lapse imaging

• Transgenic approaches: Compare wild-type plants with:

  • Plants expressing miRNA-resistant NFYA10 (NF-YA10miRres)

  • NFYA10 knockdown/knockout lines

  • Plants expressing translational fusions (pNF-YA10:GFP-NF-YA10) for protein localization

• High-throughput phenotyping: Use automated systems like ChronoRoot to quantify:

  • Lateral root angles over time

  • Differential angle formation compared to wild-type

  • Area explored by roots

• Gene expression analysis: Examine NFYA10 expression changes in response to gravitropic stimuli using qRT-PCR and reporter lines

• Target gene analysis: Investigate expression of LAZY genes (LAZY1, LAZY2, LAZY3) which are direct targets of NFYA10 and are involved in gravitropic responses

• ChIP experiments: Perform ChIP-qPCR targeting CCAAT boxes in promoters of gravitropism-related genes to identify direct regulatory targets

• Cell-specific expression: Use fluorescent reporter lines to analyze cell-type-specific expression patterns in gravity-sensing cells versus other root tissues

• Auxin transport visualization: Combine NFYA10 studies with auxin reporter lines to investigate relationships between NFYA10 regulation and auxin distribution during gravitropic responses

How can NFYA10 antibodies be used to investigate transcriptional regulation networks in plants?

For investigating NFYA10-mediated transcriptional networks in plants, implement these methodological approaches:

• ChIP-seq analysis: Perform chromatin immunoprecipitation followed by sequencing using NFYA10 antibodies to identify genome-wide binding sites

• TARGET system (Transient Assay Reporting Genome-wide Effects of Transcription factors): Use inducible NFYA10 expression combined with transcriptomic analysis to identify direct and indirect targets

• Motif analysis: Identify enriched CCAAT motifs and other cis-regulatory elements in NFYA10-bound regions

• Co-expression networks: Combine antibody-based protein expression data with transcriptomic analysis to build co-expression networks

• Transcription factor cooperation: Investigate how NFYA10 cooperates with other transcription factors by performing sequential ChIP experiments

• Cell-type specific analysis: Use fluorescence-activated cell sorting (FACS) with NFYA10 reporter lines followed by ChIP to identify cell-type-specific regulatory networks

• Developmental time series: Analyze NFYA10 binding patterns across developmental stages of lateral root formation

• Stress response networks: Examine how NFYA10 regulatory networks change in response to environmental stresses, as NF-Y transcription factors often mediate stress responses

What methodological considerations apply when studying NFYA10's regulation of LAZY genes?

When investigating NFYA10's regulation of LAZY genes, consider these methodological approaches:

• Direct binding analysis: Perform ChIP-qPCR targeting TSS-proximal CCAAT boxes in LAZY1, LAZY2, and LAZY3 promoters, using gene bodies as negative controls

• Expression correlation: Analyze expression patterns of NFYA10 and LAZY genes in different tissues and under various conditions

• Transactivation assays: Use reporter constructs containing LAZY promoters to test NFYA10-mediated activation in transient expression systems

• Mutant complementation: Test whether NFYA10 expression can rescue phenotypes in lazy mutants

• Double mutant analysis: Create and characterize nf-ya10 lazy double mutants to understand genetic interactions

• Cell-type resolution: Compare expression patterns of NFYA10 and LAZY genes at cellular resolution—while LAZY genes are highly expressed in columella cells, NFYA10 regulation may occur in endodermal cells where both are expressed

• Hormone response: Investigate how auxin and other hormones affect the NFYA10-LAZY regulatory module

• Temporal dynamics: Analyze the timing of NFYA10 expression changes relative to LAZY expression during gravitropic responses