LITAF Antibody

What is LITAF and why is it significant in immunological research?

LITAF is a transcription factor that mediates inflammatory cytokine expression, particularly in response to LPS stimulation. Research has demonstrated that LITAF plays a crucial role in regulating TNF-α production and other inflammatory mediators. Studies with LITAF-deficient mice have shown reduced expression of several cytokines including TNF-α, IL-6, sTNF-RII, and CXCL16, highlighting its importance in inflammatory responses . LITAF is primarily located in the nucleus, where it is essential for transcriptional regulation of the TNF-α gene . High expression levels of LITAF mRNA have been observed in tissues such as placenta, peripheral blood leukocytes, lymph nodes, and spleen, indicating its significance in immune function .

What types of LITAF antibodies are available for research applications?

The choice depends on your specific experimental design requirements, target species, and intended applications. Antibodies are available in both unconjugated forms and with various conjugations including HRP, fluorescent tags (FITC, PE, Alexa Fluor), and agarose for different detection methods .

What are the optimal sample preparations for detecting LITAF in different experimental contexts?

For Western blotting, LITAF is typically detected at approximately 28 kDa under reducing conditions. Validated cell lines for positive controls include A431, HeLa, HepG2, and MCF-7 human cell lines . For immunohistochemistry, human small intestine sections have been validated for LITAF detection, with optimal detection using antibody concentrations around 1.7 μg/mL overnight at 4°C . For immunofluorescence studies, the A431 human epithelial carcinoma cell line has been successfully used with antibody concentrations of approximately 10 μg/mL for 3 hours at room temperature . Proper fixation and antigen retrieval techniques are essential for maintaining epitope accessibility.

How does LITAF signaling differ from the NF-κB pathway in LPS-induced inflammatory responses?

Research with LITAF-deficient models has revealed that the LITAF signaling pathway is distinct from the NF-κB pathway, although both are triggered by LPS stimulation . Key differences include:

• Pathway activation: LITAF expression can be induced after challenge with LPS from Porphyromonas gingivalis via TLR-2 or LPS from Escherichia coli via TLR-4, both requiring functional MyD88

• Kinase dependency: The p38α MAPK specifically mediates LITAF phosphorylation and nuclear translocation, whereas the NF-κB pathway has different kinase requirements

• Inhibition profile: p38-specific inhibitors (SB203580) block LITAF nuclear translocation and reduce LPS-induced TNF-α production without completely inhibiting the NF-κB pathway

• Genetic distinction: In macrophage-specific LITAF-deficient mice, the inflammatory cytokine profile differs from that seen with NF-κB inhibition

This distinction is significant as it suggests potential for developing therapeutic strategies that selectively target specific inflammatory pathways.

What experimental approaches can distinguish between LITAF-dependent and LITAF-independent cytokine regulation?

To differentiate between LITAF-dependent and independent inflammatory pathways, researchers can employ several methodological approaches:

• Genetic manipulation studies:

  • Compare cytokine production in macLITAF−/− versus wild-type macrophages following LPS stimulation

  • Perform rescue experiments with LITAF cDNA transfection, which has been shown to restore TNF-α levels to those observed in wild-type cells

  • Use siRNA knockdown of LITAF in various cell types and measure inflammatory mediator production

• Pharmacological inhibition:

  • Apply p38α-specific inhibitors (SB203580) to block LITAF phosphorylation and nuclear translocation

  • Compare with inhibitors of other pathways (e.g., NF-κB inhibitors)

  • Assess time-course dynamics of inhibition to identify pathway-specific kinetics

• Molecular techniques:

  • Employ chromatin immunoprecipitation to identify LITAF binding sites in cytokine gene promoters

  • Use reporter assays with wild-type and mutated promoter sequences to assess LITAF-dependent transcriptional activation

  • Perform co-immunoprecipitation studies to identify LITAF interaction partners in the inflammatory response

These approaches provide complementary data to build a comprehensive understanding of LITAF's specific contributions to inflammatory responses.

How do post-translational modifications affect LITAF function and antibody recognition?

While the search results don't provide extensive details on LITAF post-translational modifications, experimental evidence indicates that:

• Phosphorylation is crucial for LITAF function:

  • p38α MAPK mediates LITAF phosphorylation in response to LPS stimulation

  • This phosphorylation appears essential for LITAF nuclear translocation and subsequent transcriptional activity

  • Researchers should consider using phospho-specific antibodies when studying active LITAF

• Impact on antibody recognition:

  • Post-translational modifications may affect epitope accessibility in different experimental contexts

  • When selecting antibodies, researchers should review the immunogen information (e.g., ABIN6262981 targets an internal region of human LITAF )

  • For studying specific LITAF activation states, antibodies raised against known phosphorylation sites would be most informative

• Experimental considerations:

  • Include phosphatase inhibitors in lysis buffers when studying phosphorylated LITAF

  • Consider native vs. denaturing conditions when assessing conformation-dependent epitopes

  • When results differ between antibodies, evaluate whether this reflects detection of different LITAF post-translational states

What are the critical parameters for successful Western blot detection of LITAF?

Optimizing Western blot detection of LITAF requires attention to several methodological details:

• Sample preparation:

  • Use reducing conditions as validated in published protocols

  • Include protease inhibitors to prevent degradation

  • LITAF appears at approximately 28 kDa on SDS-PAGE gels

• Antibody selection and dilution:

  • Primary antibody concentration: Typically 1 μg/mL for polyclonal antibodies as used in validated protocols

  • Secondary antibody selection: Must match the host species of the primary (e.g., Anti-Goat HRP for AF4695 )

  • Consider validated antibody-buffer system combinations (e.g., Immunoblot Buffer Group 1 has been used successfully with AF4695 )

• Controls and validation:

  • Positive controls: A431, HeLa, HepG2, or MCF-7 human cell lines have demonstrated detectable LITAF expression

  • Negative controls: Consider lysates from cell lines with low LITAF expression or LITAF-knockout cells

  • Specificity verification: The antibody should detect endogenous levels of total LITAF protein

Following these parameters will help ensure specific and reproducible detection of LITAF in Western blot applications.

How can researchers optimize immunohistochemical and immunofluorescence detection of LITAF?

For optimal immunohistochemical and immunofluorescence detection of LITAF:

• Tissue/cell preparation:

  • For paraffin sections: Validated protocols use paraffin-embedded sections with appropriate antigen retrieval methods

  • For cells: A431 human epithelial carcinoma cells have been successfully used with immersion fixation

• Antibody parameters:

  • For IHC: 1.7 μg/mL antibody concentration with overnight incubation at 4°C has been validated

  • For IF: 10 μg/mL antibody concentration with 3-hour room temperature incubation has shown specific staining

  • Secondary detection: For fluorescence, NorthernLights 557-conjugated Anti-Goat IgG has been validated; for chromogenic detection, HRP-DAB systems have been effective

• Visualization and counterstaining:

  • Nuclear counterstaining with DAPI helps visualize cellular context in IF applications

  • LITAF staining has been observed with specific localization to cell surfaces in some cell types

  • For tissue sections, appropriate counterstains should be selected based on the detection method

These optimized protocols have demonstrated specific LITAF detection in both cultured cells and tissue sections.

What troubleshooting approaches should be considered when LITAF detection yields unexpected results?

When encountering unexpected results with LITAF antibodies, consider these methodological troubleshooting steps:

• Weak or absent signal:

  • Verify antibody reactivity matches your experimental species (human, mouse, rat)

  • Confirm appropriate secondary antibody selection and concentration

  • Enhance antigen retrieval for fixed tissues or cells

  • Consider antibody concentration: validated concentrations range from 1-10 μg/mL depending on application

• Non-specific signals:

  • Review purification method of the antibody (e.g., peptide affinity chromatography for ABIN6262981 )

  • Increase blocking stringency (using appropriate blocking agents for the host species)

  • Consider more specific antibodies (e.g., those targeting defined epitopes like the internal region of LITAF )

  • Verify with genetic controls (LITAF knockout or knockdown samples)

• Inconsistent results between experiments:

  • Standardize lysate preparation (LITAF is detected at approximately 28 kDa under reducing conditions )

  • Document antibody lot numbers, as lot-to-lot variation can occur

  • Include validated positive controls (A431, HeLa, HepG2, or MCF-7 human cell lines )

  • Maintain consistent incubation times and temperatures across experiments

How can LITAF antibodies be used to study macrophage activation in inflammatory conditions?

LITAF antibodies enable sophisticated analysis of macrophage inflammatory responses:

• Activation dynamics studies:

  • Time-course experiments tracking LITAF expression, phosphorylation, and nuclear translocation following LPS stimulation

  • Comparative analysis between different TLR agonists (TLR-2 vs. TLR-4 pathways have both been implicated in LITAF activation )

  • Assessment of LITAF levels in different macrophage polarization states (M1 vs. M2)

• Signaling pathway analysis:

  • Co-immunoprecipitation studies to identify LITAF interaction partners (e.g., Stat6B has been identified )

  • Combined inhibitor studies examining the relationship between p38α MAPK activity and LITAF function

  • Chromatin immunoprecipitation to identify genomic targets of LITAF during inflammation

• Translational research applications:

  • Comparison of LITAF activation in healthy versus diseased tissue samples

  • Assessment of pharmacological compounds that may modulate LITAF-dependent inflammatory responses

  • Evaluation of LITAF as a biomarker for inflammatory disease progression or treatment response

These approaches provide insights into the fundamental mechanisms of inflammatory regulation and potential therapeutic targets.

What experimental models are most appropriate for studying LITAF function in inflammatory diseases?

Based on published research, several experimental models have proven valuable for studying LITAF function:

• Genetic models:

  • Macrophage-specific LITAF-deficient mice (macLITAF−/−) have demonstrated the importance of LITAF in LPS-induced inflammatory responses

  • TLR-knockout models (TLR-2−/−, TLR-4−/−, and TLR-9−/−) have helped delineate the pathways leading to LITAF activation

  • MyD88-deficient systems have shown that LITAF induction requires functional MyD88

• Cellular models:

  • Primary peritoneal macrophages provide a physiologically relevant system for studying LITAF function

  • Human cell lines (A431, HeLa, HepG2, MCF-7) express detectable levels of LITAF for mechanistic studies

  • Transfection models with wild-type or mutant LITAF constructs enable structure-function analysis

• Disease-relevant models:

  • LPS-induced endotoxemia models have shown that macLITAF−/− mice are more resistant to LPS-induced lethality

  • Various inflammatory disease models can be assessed for LITAF expression and activity using validated antibodies

  • Ex vivo analysis of patient samples can bridge basic research findings to clinical relevance

The choice of model should align with specific research questions about LITAF's role in inflammatory processes.

How can researchers distinguish between different LITAF isoforms or family members in experimental systems?

When studying LITAF, it's important to consider potential isoforms and related proteins:

• Antibody epitope considerations:

  • Select antibodies raised against specific regions of LITAF (e.g., internal region , AA 1-161 , or full recombinant protein )

  • Confirm specificity using multiple antibodies targeting different epitopes

  • Review immunogen information carefully when selecting antibodies for specific applications

• Detection methods for isoform discrimination:

  • Western blotting: Use gradient gels with extended separation times to resolve closely related isoforms

  • Immunoprecipitation followed by mass spectrometry for definitive isoform identification

  • RT-PCR with isoform-specific primers to distinguish transcript variants

• Validation approaches:

  • Compare reactivity patterns across multiple antibodies

  • Use genetic knockdown/knockout models with rescue experiments using specific isoforms

  • Consider species differences in LITAF expression and function when designing experiments

These methodological considerations help ensure that research findings are correctly attributed to specific LITAF variants.

What new approaches are emerging for studying LITAF's role in inflammatory signaling networks?

Emerging methodologies offering new insights into LITAF biology include:

• Advanced genomic and proteomic approaches:

  • ChIP-seq and CUT&RUN to map LITAF genomic binding sites with high resolution

  • Phosphoproteomics to comprehensively identify LITAF phosphorylation sites and their functional significance

  • Proximity labeling techniques (BioID, APEX) to identify context-specific LITAF interaction partners

• Single-cell analysis technologies:

  • Single-cell RNA-seq to characterize cell-specific LITAF expression patterns in heterogeneous populations

  • Single-cell proteomics to assess LITAF protein levels and modifications at individual cell resolution

  • Spatial transcriptomics to map LITAF expression in complex tissue microenvironments

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed visualization of LITAF subcellular localization

  • Live-cell imaging with fluorescently tagged LITAF to track dynamic responses to inflammatory stimuli

  • Correlative light and electron microscopy to examine LITAF in the context of cellular ultrastructure

These approaches will likely provide more nuanced understanding of LITAF's complex roles in inflammatory signaling.

How might LITAF research contribute to developing new therapeutic strategies for inflammatory diseases?

LITAF research has several potential translational applications:

• Targeted therapeutic development:

  • The distinct nature of the LITAF pathway compared to NF-κB suggests opportunities for pathway-specific anti-inflammatory approaches

  • p38α inhibitors have been shown to block LITAF nuclear translocation and reduce TNF-α production, highlighting a potential intervention point

  • Structure-based drug design targeting LITAF-specific interactions could yield novel anti-inflammatory compounds

• Biomarker development:

  • LITAF expression or activation patterns might serve as diagnostic or prognostic indicators in inflammatory conditions

  • Antibody-based assays could quantify LITAF levels or phosphorylation states in patient samples

  • Monitoring LITAF pathway activation could help assess treatment efficacy in inflammatory diseases

• Precision medicine applications:

  • Patient stratification based on LITAF pathway activation could guide personalized treatment approaches

  • Genetic variation in LITAF or its regulators might predict inflammatory disease susceptibility or progression

  • Combined modulation of LITAF and other inflammatory pathways could enable tailored therapeutic strategies

The continued development of specific LITAF antibodies and detection methods will be essential to advancing these translational opportunities.