PIL1 Antibody
Description
Applications in Research
The PIL1 antibody is employed across multiple experimental approaches to investigate Pil1’s cellular functions:
Production and Epitope Specificity
PIL1 antibodies are generated using recombinant protein strategies:
Key Notes:
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Antibodies raised against GST-Pil1 fusion proteins enable detection of Pil1 in both yeast and heterologous systems (e.g., COS-7 cells) .
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Cross-reactivity with Lsp1 complicates interpretation in eisosome studies but highlights structural similarities .
Mitochondrial Regulation
PIL1 antibodies revealed Pil1’s role in mitochondrial fission and mitophagy:
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Mitochondrial Morphology: pil1Δ cells exhibit abnormal fusion/aggregation, while overexpression causes fragmentation .
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Mitophagy Defects: PIL1 deletion reduces mitophagy and bulk autophagy, elevating ROS and protein aggregates .
Eisosome and Membrane Organization
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Cortical Filaments: Pil1 forms stable cortical structures critical for membrane organization. Overexpression disrupts cytokinesis and polarity in Schizosaccharomyces pombe .
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Membrane Binding: Pil1 preferentially binds PI(4,5)P₂-containing membranes, forming tubular scaffolds in vitro .
Phosphorylation and Function
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Pkh-Kinase Phosphorylation: Pil1 phosphorylation shifts its assembly equilibrium, modulating eisosome density and localization .
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Functional Implications: Phosphorylated Pil1 variants exhibit altered membrane-binding kinetics and eisosome dynamics .
Table 1: PIL1 Antibody Applications
| Application | Technique | Outcome | Model Organism |
|---|---|---|---|
| Mitochondrial localization | Trypsin digestion + immunoblotting | Pil1 localized to outer mitochondrial membrane | Saccharomyces cerevisiae |
| Membrane binding | Liposome cosedimentation | PI(4,5)P₂ preference confirmed | In vitro |
| Autophagy studies | GFP-Atg8 processing assay | Reduced autophagy in pil1Δ | S. cerevisiae |
Table 2: Phosphorylation Sites Detected via PIL1 Antibody
| Site | Residue | Method | Functional Impact |
|---|---|---|---|
| 1 | Ser16 | Tandem mass spectrometry | Modulates eisosome assembly |
| 2 | Ser45 | NBD-fluorophore coupling | Critical for membrane binding |
| 3 | Thr233 | Tandem mass spectrometry | Phosphorylation by Pkh-kinases |
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- PIL1 acts as a decelerator of growth during the early stages of shade avoidance. PMID: 24895419
- PIL1 is a direct target of phytochrome signaling and plays a critical role in the response to long-term shade in Arabidopsis. PMID: 16565297
- PIL1 is essential for the rapid light-induced expression of the PSEUDO-RESPONSE REGULATOR9 (APRR9) gene, suggesting a regulatory function within the early phytochrome-induced transcriptional network. PMID: 16891401
Q&A
Basic Research Questions
How is PIL1 antibody validated for specificity in yeast studies?
PIL1 antibodies are typically validated using:
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Western blotting: Compare protein extracts from wild-type (WT) and pil1Δ strains. PIL1 antibodies should detect a band in WT (~40 kDa) but not in knockout strains .
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Immunofluorescence: Verify cortical eisosome localization in WT cells and absence of signal in pil1Δ mutants .
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Functional rescue: Overexpress PIL1 in pil1Δ strains and confirm restored mitochondrial morphology or autophagy activity .
What methods are optimal for detecting PIL1-protein interactions?
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Co-immunoprecipitation (Co-IP): Use crosslinkers like DSP to stabilize transient interactions. PIL1 interacts with mitochondrial fission proteins (e.g., Fis1/Dnm1), which can be confirmed via immunoblotting after IP .
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Yeast two-hybrid: Screen for direct interactions (e.g., PIL1-Fis1 binding) .
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Proximity ligation assays: Visualize PIL1’s spatial relationship with membrane scaffolds in live cells .
Advanced Research Challenges
How to resolve contradictory PIL1 localization data under overexpression?
PIL1 forms cytoplasmic rods when overexpressed, confounding cortical eisosome studies . To address this:
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Titrate expression levels: Use weak promoters (e.g., TEF1) instead of strong inducible systems.
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Combine localization markers: Co-stain with mitochondrial (e.g., Om45-GFP) or cortical markers (e.g., Lsp1-mCherry) to distinguish mislocalized PIL1 .
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Validate functionally: Assess mitochondrial fragmentation (a PIL1-dependent phenotype) to confirm physiological relevance .
How to analyze PIL1’s role in autophagy vs. mitochondrial dynamics?
What controls are critical when studying PIL1-associated phosphorylation?
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Phosphoproteomics: Include rts1Δ (PP2A regulatory subunit) mutants to identify phosphatase-dependent phosphorylation sites .
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Isotopic labeling: Use dimethyl labeling (e.g., heavy/medium/light) to quantify phosphorylation changes in PIL1-EGFP vs. untagged strains .
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Kinase inhibition: Treat cells with TORC2 inhibitors (e.g., KU-0063794) to dissect signaling pathways regulating PIL1 .
Methodological Optimization
How to mitigate PIL1 aggregation in vitro?
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Buffer optimization: Use PBS with 150 mM potassium acetate and 10% glycerol to stabilize PIL1 during purification .
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Detergent screening: Test CHAPS or digitonin (0.1–0.5%) to solubilize PIL1 without disrupting BAR domain-membrane interactions .
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Negative-stain EM: Confirm filament integrity post-purification (e.g., ~10 nm diameter filaments) .
How to reconcile conflicting mitophagy data in fis1Δ vs. dnm1Δ strains?
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Genetic epistasis: Compare fis1Δpil1Δ and dnm1Δpil1Δ double mutants. Mitophagy is abolished in dnm1Δpil1Δ but partially retained in fis1Δpil1Δ .
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Overexpression rescue: Express PIL1 in fis1Δpil1Δ to restore mitophagy (e.g., 60% recovery) .
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Pathway-specific reporters: Use GFP-Atg32 (mitophagy-specific) vs. GFP-Atg8 (general autophagy) to isolate PIL1’s roles .
Data Interpretation
Why does PIL1 overexpression cause cytotoxicity in fission yeast?
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Mechanism: Excess PIL1 forms cytoplasmic rods (Fig 8A ), displacing cortical polarity factors (e.g., Tea1, Cdr2).
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Validation: Perform FRAP on PIL1-GFP rods; minimal recovery (<20% in 10 min) confirms stable aggregates .
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Mitigation: Use inducible promoters (e.g., nmt41) and monitor growth rates to avoid overexpression artifacts .
