CML38 Antibody
Description
Functional Roles in Hypoxia and Stress Adaptation
CML38 integrates calcium signaling with cellular responses to low oxygen:
Hypoxia-Induced Stress Granule Formation
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SG assembly: CML38 co-localizes with stress granule markers (e.g., RBP47B) and RNA-processing proteins during hypoxia .
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mRNA regulation: Associates with arrested preinitiation complexes and inhibits translation, conserving energy under stress .
Autophagy Regulation via SGS3 and CDC48
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SGS3 interaction: Directly binds SGS3, a key RNA-binding protein in siRNA biogenesis, promoting SGS3 granule turnover during extended hypoxia .
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CDC48 dependency: Interacts with the ubiquitin segregase CDC48A, essential for autophagic degradation of SGS3 bodies .
Table 2: CML38-Dependent Autophagy Pathways
| Component | Role in Autophagy | Outcome in cml38 Mutants |
|---|---|---|
| SGS3 | RNA-binding protein in siRNA biogenesis | Accumulation, reduced turnover |
| CDC48A | AAA+ ATPase for protein quality control | Disrupted SGS3 granule degradation |
| Reoxygenation | Calcium-dependent SG breakdown post-hypoxia | Sustained autophagosome accumulation |
Genetic and Biochemical Validation
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Hypoxia sensitivity: cml38 mutants show reduced survival, stunted growth, and impaired root elongation under low oxygen .
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Rescue experiments: Transgenic CML38 genomic fragments restore wild-type phenotypes in cml38 mutants .
Protein Interaction Networks
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BACTH analysis: Confirmed direct interactions with SGS3, CDC48A, and DUF581-5 but not GRP7, GRP8, or eIF4A .
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Immunoprecipitation: Co-purified with RNA splicing factors (GRP7/8) and translation machinery (eIF4A), suggesting indirect associations in stress granules .
Functional Analogy to Viral Suppressors
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PTGS suppression: Like HC-Pro (Turnip Mosaic Virus), CML38 inhibits RNA silencing by targeting SGS3 and RDR6-dependent pathways, reducing secondary siRNA production .
Unresolved Questions and Future Directions
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Calcium dependency: How calcium influx during hypoxia triggers CML38 granule formation remains unclear.
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Evolutionary conservation: Potential homologs in other plants or organisms have not been systematically explored.
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Therapeutic relevance: While unrelated to CD38 monoclonal antibodies (e.g., daratumumab for myeloma), CML38’s role in RNA granule dynamics may inform plant stress adaptation strategies.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- CML38 functions as a core hypoxia response calcium sensor protein. PMID: 26634999
Q&A
Experimental Design for Studying CML38 Function
Q: How can I design an experiment to study the function of CML38 in plant stress responses using the CML38 antibody? A: To study CML38's role in plant stress responses, you can use a combination of molecular biology and cell biology techniques. First, generate transgenic plants expressing CML38 fused to a fluorescent protein (e.g., YFP) to visualize its localization under stress conditions. Use immunoprecipitation followed by mass spectrometry to identify interacting proteins, which can provide insights into its function in stress granules . Additionally, employ quantitative reverse transcription PCR (qRT-PCR) to monitor changes in CML38 expression levels under different stress conditions.
Antibody Validation and Specificity
Q: How do I validate the specificity of the CML38 antibody for use in immunoprecipitation and Western blotting? A: Validate the specificity of the CML38 antibody by performing Western blotting on extracts from wild-type and CML38 knockout plants. The antibody should detect a band corresponding to CML38 in wild-type but not in knockout plants. Additionally, use immunoprecipitation followed by mass spectrometry to confirm that the antibody specifically pulls down CML38 and associated proteins .
Data Analysis and Interpretation
Q: How can I analyze and interpret data from experiments using the CML38 antibody to study stress responses in plants? A: Analyze data by comparing the expression levels of CML38 and associated proteins under different stress conditions (e.g., hypoxia) using statistical methods. Interpretation should focus on how changes in CML38 expression correlate with stress responses, such as the formation of stress granules and changes in plant survival rates . Consider using bioinformatics tools to analyze mass spectrometry data for protein interactions.
Advanced Research Questions: Mechanistic Insights
Q: What are some advanced research questions related to CML38 that could provide mechanistic insights into plant stress responses? A: Advanced research questions might include:
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Mechanism of CML38 localization: How does CML38 localize to stress granules, and what role does calcium play in this process?
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Protein interactions: What are the specific interactions between CML38 and other proteins in stress granules, and how do these interactions modulate stress responses?
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Signaling pathways: How does CML38 integrate into broader signaling pathways to regulate plant stress responses?
Contradictory Data Analysis
Q: How can I address contradictory data when studying CML38's role in plant stress responses? A: Address contradictory data by carefully reviewing experimental conditions, ensuring consistency in methods, and considering potential variables that might influence results. Use statistical analysis to assess the significance of observed differences. If discrepancies persist, consider additional experiments to validate findings or explore alternative explanations for the observed phenomena.
Methodological Considerations for CML38 Antibody Use
Q: What methodological considerations should I keep in mind when using the CML38 antibody for immunoprecipitation and Western blotting? A: Key considerations include:
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Optimization of antibody concentration: Ensure the optimal concentration of the CML38 antibody is used to avoid non-specific binding.
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Control experiments: Include appropriate controls, such as using extracts from knockout plants, to validate specificity.
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Buffer conditions: Optimize buffer conditions for immunoprecipitation to maintain protein interactions.
Data Presentation and Visualization
Q: How can I effectively present and visualize data from experiments using the CML38 antibody? A: Use clear and concise figures to present data, such as bar graphs for qRT-PCR results and Western blots to show protein expression. For protein interactions, consider using network diagrams to visualize mass spectrometry data. Ensure that all figures are well-labeled and include appropriate controls for comparison.
Example Data Table: CML38 Expression Under Hypoxia
| Time (h) | CML38 Expression (Fold Change) |
|---|---|
| 0 | 1 |
| 3 | 50 |
| 6 | 300 |
This table illustrates the rapid increase in CML38 expression in response to hypoxia, which is crucial for understanding its role in stress responses .
