AHL10 Antibody
Description
AHL10 Protein Overview
AHL10 belongs to the Clade B AHL family (AHL1–AHL14) and contains an N-terminal AT-hook DNA-binding domain and a variable C-terminal region involved in protein-protein interactions . It regulates chromatin association of downstream targets like RRP6L1 (RNA recognition motif-containing protein 6-like 1) during drought stress .
Functional Role in Stress Responses
AHL10 phosphorylation at residues S313/S314 determines its activity during low water potential (ψw) stress:
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Phosphomimetic mutations (e.g., S314D) disrupt nuclear foci formation and enhance growth suppression under drought .
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Phosphorylation dynamics: Controlled by MAP kinases (MPK3/MPK6) and counteracted by the phosphatase HAI1 .
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Transcriptional regulation: AHL10 modulates stress-responsive genes, including AT-rich transposons (At5g35935) and jasmonic acid biosynthesis pathways .
Interaction Partners and Complex Formation
AHL10 forms hetero-complexes with other AHLs (AHL1, AHL3, AHL13) and interacts with:
Antibodies Used in AHL10 Research
While no AHL10-specific antibody is detailed, studies employed epitope tags and cross-reactive antibodies:
Key Research Findings
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Phosphorylation-dependent chromatin binding: AHL10 phosphorylation at S313/S314 does not affect DNA binding but modulates RRP6L1 association .
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Stress-specific regulation: AHL10 and AHL13, despite 72% sequence similarity, have non-redundant roles due to hetero-complex formation .
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Transcriptome impact: ahl10-1 mutants show dysregulation of 40 stress- and development-related genes, including At5g35935 (transposon) and At5g35940 (downstream gene) .
Methodological Insights
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Yeast two-hybrid screening: Identified AHL10 interactors like RRP6L1 and CDC20.2 .
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Chromatin immunoprecipitation (ChIP): Revealed AHL10 binding to AT-rich regions adjacent to stress-responsive genes .
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Phosphoproteomics: MPK6 phosphorylates AHL10 S314, while HAI1 dephosphorylates it to attenuate drought responses .
Implications for Plant Biology
AHL10 exemplifies how post-translational modifications fine-tune stress adaptation:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
KEGG: ath:AT2G33620
UniGene: At.48546
Q&A
Basic Research Questions
What are the primary research applications of AHL10 antibodies in plant molecular studies?
AHL10 antibodies are primarily used to investigate the protein’s role in transcriptional regulation under abiotic stress, particularly salt stress. Key applications include:
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Chromatin immunoprecipitation (ChIP-seq) to map genomic binding sites (e.g., identifying 12,803 AHL10-associated genes with promoter-region enrichment) .
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Phosphorylation state analysis using Phos-tag assays to study post-translational modifications (e.g., detecting salt-induced phosphorylation at Ser314) .
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Protein-protein interaction studies (e.g., co-immunoprecipitation with SUVH2/9 histone methyltransferases) .
How is AHL10 antibody specificity validated in experimental systems?
Validation typically involves:
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Genetic controls: Comparing wild-type and AHL10 knockout lines (e.g., AHL10-GFP/ahl10 mutants) to confirm signal loss in knockouts .
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Epitope tagging: Using transgenic lines expressing GFP- or MYC-tagged AHL10 for comparative western blotting .
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Cross-reactivity tests: Ensuring no binding to paralogs like AHL2 or AHL18 via immunoblots .
Advanced Experimental Design
How do researchers address contradictory data on AHL10’s stress-response mechanisms?
Discrepancies in AHL10’s role (e.g., transcriptional activation vs. repression) are resolved by:
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Context-specific phosphorylation analysis: Testing Ser314 phosphorylation under varying NaCl concentrations (0–150 mM) .
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Combinatorial mutant studies: Using AHL10 S314A/S317A phospho-null variants to isolate CDK8-dependent effects .
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Time-course ChIP-seq: Monitoring dynamic chromatin occupancy during stress progression .
What methodologies optimize AHL10 antibody performance in low-abundance scenarios?
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Signal amplification: Tyramide-based systems for immunohistochemistry in root tissues.
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Pre-clearing lysates: Reducing non-specific binding using AHL10-GFP/cdk8 mutant extracts .
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Multiplexed IP-MS: Coupling immunoprecipitation with mass spectrometry to detect weak interactors .
Data Interpretation Challenges
How are AHL10 antibody-derived ChIP-seq peaks distinguished from background noise?
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Replicate concordance: Requiring overlapping peaks across ≥2 biological replicates (e.g., 14,584 vs. 16,657 peaks in two runs) .
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Motif enrichment analysis: Filtering peaks lacking AT-rich sequences (e.g., (G/A)ATTTT(A/T)A motifs) .
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Functional validation: Correlating binding sites with RNA-seq data from AHL10 mutants .
What controls are critical for phospho-specific AHL10 antibody experiments?
Methodological Recommendations
Which techniques complement AHL10 antibody studies in chromatin biology?
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Electrophoretic mobility shift assays (EMSAs): Validate AHL10’s binding to AT-rich MAR sequences (e.g., DREB2A promoter probes) .
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Luciferase reporter systems: Quantify transcriptional repression via AHL10-SUVH2/9 recruitment (e.g., 50% reduction in MYB15 LUC/REN ratios) .
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Structured illumination microscopy: Resolve nuclear AHL10 foci formation (diameter: 200–400 nm) .
How is batch-to-batch antibody variability mitigated in long-term studies?
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Aliquot normalization: Pre-testing 3–5 aliquots across biological replicates (CV < 15%).
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Cross-platform validation: Confirming results with independent methods (e.g., AHL10 CRISPR lines vs. antibody-based detection) .
Technical Troubleshooting
Why might AHL10 antibody signals persist in cdk8 mutant lines under salt stress?
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Compensatory phosphorylation: Residual activity from related kinases (e.g., MAPKs) at Ser313/317 .
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Epitope masking: Conformational changes in non-phosphorylated AHL10 reducing antibody accessibility .
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Solution: Combine genetic (cdk8 mutants) and pharmacological (CDK8 inhibitor IV) approaches .
