TOLLIP Antibody
Description
Introduction to TOLLIP Antibody
TOLLIP (Toll-Interacting Protein) antibodies are essential tools for studying the multifaceted roles of TOLLIP, a critical adaptor protein involved in innate immunity, intracellular trafficking, and inflammatory regulation. These antibodies enable researchers to detect TOLLIP expression, assess its interactions with signaling molecules like IRAK and STAT3, and investigate its regulatory effects on pathways such as TLR/IL-1R signaling and selective autophagy .
Key Research Findings on TOLLIP
Immune Regulation
-
Negative Regulation of TLR/IL-1R Signaling: TOLLIP suppresses NF-κB and MAPK pathways by inhibiting IRAK-1 kinase activity and promoting IRAK-1 degradation. This dampens proinflammatory cytokine production (e.g., TNF-α, IL-6) in response to IL-1β and LPS .
-
Modulation of STAT3: TOLLIP interacts with STAT3 to inhibit IL-13-induced eotaxin-3 production in airway epithelial cells, highlighting its role in mitigating type 2 inflammatory responses in asthma .
Intracellular Trafficking and Autophagy
-
ER Protein Clearance: TOLLIP acts as a cargo adaptor for lysosomal degradation of misfolded ER membrane proteins (e.g., mutant VAPB and Seipin), reducing ER stress in neurodegenerative disease models .
-
TGF-β Receptor Regulation: TOLLIP cooperates with Smad7 to promote ubiquitination and degradation of TGF-β receptors, thereby suppressing TGF-β signaling .
Disease Associations
-
Tuberculosis Susceptibility: TOLLIP polymorphisms (rs3750920, rs5743899) correlate with reduced mRNA expression and increased IL-6/TNF production, elevating TB risk in human cohorts .
-
Triple-Negative Breast Cancer: TOLLIP-mediated autophagy pathways are implicated in cancer progression, with TOLLIP depletion accelerating tumor growth .
Applications of TOLLIP Antibodies
TOLLIP antibodies are validated for diverse experimental applications:
Protocols and Technical Considerations
-
Recommended Dilutions:
-
Buffer Compatibility: Most antibodies are stable in PBS with 0.02% sodium azide and 50% glycerol .
Recent Advances and Clinical Implications
-
SARS-CoV-2: TOLLIP-mediated selective autophagy degrades ACE2, potentially modulating COVID-19 severity .
-
Neurodegeneration: TOLLIP clears mutant proteins linked to ALS and hereditary spastic paraplegia, offering therapeutic targets .
-
Asthma: TOLLIP deficiency exacerbates IL-13-driven STAT3 activation, suggesting STAT3 inhibitors as potential treatments .
Product Specs
Q&A
How should researchers validate TOLLIP antibody specificity in a new experimental system?
Validation requires a multi-step approach:
-
Molecular Weight Verification: Perform WB using lysates from cells/tissues with confirmed TOLLIP expression (e.g., HepG2, HEK-293). The observed band should align with the predicted 30 kDa size . Discrepancies may indicate splice variants or post-translational modifications.
-
Knockout Controls: Use TOLLIP-knockout (KO) cell lines or tissues to confirm absence of signal. For example, TOLLIP-KO mice show abolished staining in vascular smooth muscle cells (VSMCs) .
-
Cross-Reactivity Checks: Test antibody reactivity across species (human, mouse, rat) using tissues like brain (mouse/rat) or liver (human) .
-
Orthogonal Validation: Compare results with alternative methods (e.g., IF/ICC in U2OS cells vs. flow cytometry in the same ).
Table 1: Recommended Validation Workflow
| Step | Method | Positive Control | Negative Control |
|---|---|---|---|
| 1 | WB | HepG2 lysate | TOLLIP-KO lysate |
| 2 | IHC | Human glioma tissue | Knockout tissue |
| 3 | IF/ICC | U2OS cells | siRNA-treated cells |
What are optimal dilution protocols for TOLLIP antibodies in different applications?
Dilution ranges vary by experimental context:
-
WB: Start at 1:5,000 for high-abundance samples (e.g., brain tissue) . For low-expression systems (e.g., VSMCs), increase to 1:50,000 .
-
IHC: Use 1:50–1:500 with antigen retrieval (TE buffer pH 9.0) .
-
IF/ICC: 1:50–1:800, adjusting based on fixation method (e.g., paraformaldehyde vs. methanol) .
Table 2: Dilution Guidelines Across Systems
| Application | Tissue/Cell Type | Dilution | Antigen Retrieval |
|---|---|---|---|
| WB | Mouse brain | 1:5,000 | Not required |
| IHC | Human glioma | 1:200 | TE buffer, pH 9.0 |
| IF/ICC | HepG2 | 1:200 | 0.1% Triton X-100 |
How can TOLLIP antibody data resolve contradictions in TLR pathway activation studies?
Conflicting reports on TOLLIP’s role in TLR signaling often arise from model-specific variables:
-
Cell Type: TOLLIP suppresses IRAK1 in HEK-293 cells but enhances Pseudomonas aeruginosa clearance in alveolar epithelial cells .
-
Stimulation Context: Prolonged LPS exposure upregulates TOLLIP in nasal epithelium, dampening TNF-α production , whereas acute VSMC injury downregulates TOLLIP, activating Akt signaling .
-
Solution: Perform time-course experiments with pathway inhibitors (e.g., Akt inhibitor MK-2206) and compare TOLLIP localization (cytosolic vs. membrane-bound) .
What strategies improve TOLLIP detection in autophagy studies?
TOLLIP’s role in autophagic clearance of protein aggregates requires precise detection:
-
Fixation Adjustments: Use methanol-free fixatives (e.g., 4% PFA) to preserve epitopes for IF/ICC .
-
Co-staining Markers: Pair TOLLIP with LC3B (autophagosome marker) and p62/SQSTM1 in HepG2 cells .
-
Pharmacological Perturbation: Treat cells with bafilomycin A1 to block autophagic flux and quantify TOLLIP-positive puncta .
How do genetic models clarify TOLLIP’s dual roles in inflammation and proliferation?
-
KO Models: Global TOLLIP-KO mice exhibit exacerbated neointima formation post-vascular injury due to unregulated VSMC proliferation .
-
Transgenic Overexpression: SMC-specific TOLLIP-TG mice show reduced intimal hyperplasia (I/M ratio: 0.2 vs. 0.7 in wild-type) .
-
Mechanistic Insight: Rescue experiments with Akt inhibitors (e.g., AKTI) in KO models restore proliferation control, confirming TOLLIP-Akt crosstalk .
Methodological Recommendations for Contradictory Data
-
Contextualize Reactivity: TOLLIP antibodies may detect isoforms in brain (30 kDa) vs. modified forms in cancer cells (e.g., phosphorylated TOLLIP at 32 kDa) .
-
Quantitative Normalization: Use housekeeping proteins (e.g., GAPDH) validated in the same matrix (e.g., nuclear vs. cytoplasmic fractions) .
-
Cross-Validation: Compare datasets from multiple clones (e.g., 11315-1-AP vs. 84711-5-RR) .
Product Science Overview
Toll-interacting protein (TOLLIP) is a ubiquitously expressed intracellular adaptor protein that plays a crucial role in various intracellular signaling pathways. It is involved in mediating inflammatory responses, promoting autophagy, and enabling vacuole transport within cells . TOLLIP is increasingly recognized for its role in the pathophysiology of several diseases, including neurodegenerative diseases, pulmonary diseases, cardiovascular diseases, inflammatory bowel disease, and malignancies .
TOLLIP is a ubiquitin-binding protein that interacts with several components of the Toll-like receptor (TLR) signaling cascade . It acts as a negative regulator of pro-inflammatory responses, particularly in the context of viral infections such as influenza A virus (IAV) . TOLLIP’s ability to modulate inflammatory signaling is critical for maintaining cellular homeostasis and preventing excessive inflammation.
TOLLIP is a key player in the regulation of inflammatory responses. It modulates the IL-1R/TLR-NF-κB signaling pathways, which are essential for the body’s immune response to infections . In the central nervous system, TOLLIP has been shown to play a role in neuroinflammation, particularly in the substantia nigra, a region affected in Parkinson’s disease . Studies have demonstrated that TOLLIP deficiency can lead to exaggerated inflammatory responses and increased susceptibility to neuroinflammation .
TOLLIP is also involved in promoting autophagy, a cellular process that degrades and recycles damaged cellular components . This function is vital for maintaining cellular health and preventing the accumulation of damaged proteins and organelles. Additionally, TOLLIP plays a role in vacuole trafficking, which is essential for the transport of cellular materials and the maintenance of cellular homeostasis .
Research has shown that TOLLIP is implicated in the pathogenesis of various diseases. For example, genetic variations in TOLLIP have been associated with reduced airway epithelial expression and poor lung function in asthma patients . In neurodegenerative diseases like Parkinson’s disease, TOLLIP’s role in modulating neuroinflammation suggests it could be a potential target for neuroprotective therapies . Furthermore, TOLLIP’s involvement in autophagy and vacuole trafficking indicates its potential role in other conditions related to these processes .
