CLT1 Antibody
Description
Mechanism of Action
CLT1 exerts cytotoxicity through a multi-step process:
Target Binding and Internalization
Downstream Effects
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Autophagic cell death: Lysosomal leakage triggers LC3-mediated autophagy
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Unfolded protein response (UPR): ER stress pathways activated via fibronectin misfolding
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Gene regulation: Upregulation of stress-response genes (e.g., HSPA5, DDIT3) observed in treated endothelial cells
In Vitro Cytotoxicity
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Cell lines affected: Bladder (J82, UMUC3), kidney (786-0), breast (MCF7), and lung (A549) cancers
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Dose response: EC50 ≈ 75 µg/ml in bladder cancer cells with fibronectin
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Synergy: Activity enhanced 3–5× by fibronectin but inhibited by fibrinogen
In Vivo Efficacy
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Angiogenesis inhibition: Reduced tumor vascularization in murine models
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Tumor growth suppression: 40–60% reduction in bladder cancer xenograft volume
Biomarker Correlations
Diagnostic Potential
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Tumor homing: Fluorescein-labeled CLT1 selectively binds bladder cancer tissue ex vivo (94% specificity)
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Imaging: Co-localizes with CLIC3 and fibronectin in angiogenic vasculature
Therapeutic Advantages
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Selectivity: Non-toxic to mature endothelial cells or normal bladder epithelium
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Resistance profile: Mechanistically distinct from BCG immunotherapy and mitomycin C
Limitations and Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Basic Research Questions
How to validate CLT1 antibody specificity in endothelial and tumor cell models?
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Perform dual-label immunofluorescence (IF) with markers like CD31 (endothelial cells) or tumor-specific integrins (α5β1/αvβ3). Use siRNA knockdown of CLTCL1 to confirm signal loss .
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Validate cross-reactivity by comparing staining patterns in CLTC-expressing vs. CLTCL1-expressing cell lines (e.g., HeLa vs. CLTC-knockout models) .
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Include blocking controls with immunogen peptides (e.g., 10 µg/mL CLT1 peptide) to assess nonspecific binding .
What experimental conditions optimize CLT1 antibody performance in subcellular localization studies?
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Use fresh-frozen tissue sections fixed with acetone (avoid paraformaldehyde for membrane protein preservation) .
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For lysosomal co-localization, pre-treat cells with 100 nM Lysotracker Red for 1 hr before fixation .
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Optimize dilution ranges (IF/ICC: 1:50–1:500) and buffer additives (0.5% BSA reduces background) .
How to design dose-response experiments for CLT1 cytotoxicity assays?
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Titrate CLT1 between 10–100 µg/mL with constant fibronectin (10 µg/mL) .
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Include fibrinogen (140 µg/mL) as a negative control to block CLT1-fibronectin aggregation .
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Measure cell viability via propidium iodide staining and flow cytometry (15,000 events/sample) .
Advanced Research Questions
How to resolve contradictory CLT1 cytotoxicity data across tumor types?
| Tumor Type | Integrin α5β1 Expression | CLIC3 Expression | CLT1 IC₅₀ (µg/mL) |
|---|---|---|---|
| Bladder (J82) | High | High | 75 |
| Kidney (786-0) | Low | Low | >100 |
| Prostate (DU145) | Moderate | Moderate | 85 |
What methodologies distinguish CLT1-induced autophagic cell death from apoptosis?
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Monitor LC3-I/LC3-II conversion via Western blot (4–12% Bis-Tris gels) .
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Inhibit autophagy with 10 nM bafilomycin A1 and compare cytotoxicity (reduction indicates autophagy dependency) .
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Assess lysosomal leakage by measuring cytoplasmic cathepsin D levels (Subcellular Protein Fractionation Kit) .
How to analyze CLT1-fibronectin co-aggregation dynamics in live-cell imaging?
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Use fluorescein-conjugated CLT1 (25 µg/mL) with time-lapse confocal microscopy (5-min intervals for 24 hrs) .
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Block RGD-dependent binding with GRGDSP peptide (100 µM) to confirm integrin-mediated uptake .
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Quantify co-localization with fibronectin using Manders’ coefficient (ImageJ plugins) .
Methodological Considerations for Data Reproducibility
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Fibronectin source variability: Plasma fibronectin (10 µg/mL) enhances CLT1 activity vs. cellular fibronectin .
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Urine interference: Include 50% urine in bladder cancer assays to mimic physiological conditions .
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Storage protocols: Aliquot CLT1 antibody in PBS + 50% glycerol, store at -20°C (prevents freeze-thaw degradation) .
