PELI1 Antibody

What is PELI1 and what are its primary functions?

PELI1 (Pellino homolog 1) is an E3 ubiquitin-protein ligase that plays critical roles in immune regulation. Its primary functions include negatively regulating T-cell activation and preventing autoimmunity by mediating K48 ubiquitination of nuclear c-Rel, a member of the NF-κB family of transcription factors . PELI1 is abundantly expressed in lymphocytes, particularly T cells, and its expression is induced during T cell activation . Additionally, PELI1 functions as a negative regulator of noncanonical NF-κB signaling, which helps restrain the pathogenesis of lupus-like autoimmune diseases . Research has demonstrated that PELI1 associates with NF-κB inducing kinase (NIK) and mediates its Lys48 ubiquitination and degradation, thereby inhibiting excessive immune responses .

What experimental models have been used to study PELI1 function?

Several experimental models have been employed to investigate PELI1 function. The most common model is the PELI1 knockout (PELI1−/−) mouse, which displays spontaneous autoimmunity characterized by multiorgan inflammation and autoantibody production . Researchers have also utilized bone marrow (BM) chimeric models in which lethally irradiated Rag1−/− mice are reconstituted with mixed BM cells from different sources, such as SJL mice and wild-type or PELI1-KO mice . Another valuable model is the bm12-induced lupus-like disease model, where recipient mice receive adoptive transfer of wild-type or PELI1-deficient CD4+ T cells, enabling the study of autoimmunity development . Additionally, the H1N1 influenza virus infection model has been used to examine how PELI1 regulates Tfh-mediated humoral immunity and viral clearance .

Which antibody applications are validated for PELI1 detection?

PELI1 antibodies have been validated for multiple research applications. Western blot (WB) analysis is commonly used to detect PELI1 protein expression, with recommended dilutions of 1:500-1:1000 . Immunoprecipitation (IP) is another validated application, requiring 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Immunohistochemistry (IHC) can be performed at dilutions of 1:50-1:500, with antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0) . The antibody has been tested positive in THP-1 cells for WB, SH-SY5Y cells for IP, and mouse brain tissue for IHC . Additionally, the antibody has shown reactivity with human, mouse, and rat samples, making it versatile for cross-species research .

How does PELI1 deficiency affect T-cell function?

PELI1 deficiency results in hyper-activation of T cells, rendering them refractory to suppression by T regulatory cells and transforming growth factor-β (TGF-β) . In PELI1 knockout mice, CD4+ T cells produce significantly higher levels of the major T cell cytokines IL-2 and interferon-γ (IFN-γ) in response to CD3-CD28 stimulation . This phenotype is even more pronounced in PELI1−/− CD8+ T cells, which display markedly enhanced proliferation ability . Notably, PELI1-deficient T cells can respond to TCR stimulation even in the absence of CD28 co-stimulation, suggesting that PELI1 is crucial for maintaining proper T cell activation thresholds . Additionally, PELI1 deficiency enhances T follicular helper (Tfh) cell differentiation, promoting increased germinal center B cell formation and antibody production .

How does PELI1 regulate NF-κB signaling at the molecular level?

PELI1 exerts complex regulatory control over NF-κB signaling through distinct mechanisms. For canonical NF-κB signaling in T cells, PELI1 does not affect the activation of IKK (inhibitor of nuclear factor kappa B kinase) but specifically targets nuclear c-Rel for K48 ubiquitination, leading to its degradation and limiting sustained T cell activation . This selective negative regulation is critical for maintaining peripheral T-cell tolerance. In the noncanonical NF-κB pathway, PELI1 functions as an E3 ligase that associates directly with NF-κB inducing kinase (NIK) to mediate its Lys48 ubiquitination and subsequent degradation . Through this mechanism, PELI1 prevents excessive noncanonical NF-κB activation in B cells, which would otherwise lead to uncontrolled plasma cell differentiation and autoantibody production . These distinct regulatory roles highlight PELI1's importance as a checkpoint molecule in multiple immune cell types.

What is the relationship between PELI1 and autoimmune diseases?

PELI1 functions as a critical protective factor against autoimmune diseases through multiple mechanisms. In human patients, PELI1 levels negatively correlate with disease severity in systemic lupus erythematosus (SLE), suggesting its potential as a biomarker for disease progression . Mechanistically, PELI1 deficiency promotes autoimmunity in several ways. In T cells, loss of PELI1 leads to accumulation of nuclear c-Rel, resulting in hyperresponsiveness to stimulation and resistance to regulatory T cell suppression . PELI1-deficient mice spontaneously develop multiorgan inflammation, produce autoantibodies, and show immune complex deposition in kidney glomeruli, all hallmarks of lupus-like disease . In B cells, PELI1 deficiency enhances noncanonical NF-κB signaling, promoting excessive plasma cell differentiation and autoantibody production . Additionally, PELI1 suppresses T follicular helper (Tfh) cell differentiation, and its deficiency accelerates lupus-like disease in the bm12 adoptive transfer model . These findings establish PELI1 as a multifaceted regulator of autoimmunity.

How does PELI1 influence T follicular helper (Tfh) cell-mediated immunity?

PELI1 functions as an intrinsic negative regulator of T follicular helper (Tfh) cell differentiation and function. In PELI1-deficient mice, the frequency of Tfh cells is significantly increased following immunization compared to wild-type mice . Mechanistically, PELI1 inhibits inducible T-cell co-stimulator (ICOS) expression, which is crucial for Tfh cell development . When PELI1-deficient CD4+ T cells are transferred into bm12 mice, they differentiate into Tfh cells at a higher rate than wild-type cells, inducing robust increases in germinal center B cells and plasma cells . This enhanced Tfh differentiation leads to increased production of serum anti-nuclear antibodies, anti-dsDNA, anti-ssDNA, and anti-histone IgG, accelerating lupus-like autoimmunity . Interestingly, this phenomenon has beneficial effects in the context of viral infection. In H1N1 influenza virus infection models, PELI1-deficient CD4+ T cells promote stronger Tfh-mediated humoral immunity, higher antibody production in bronchoalveolar lavage fluid and serum, and more efficient viral clearance . These findings demonstrate PELI1's dual role in regulating Tfh cells – protective against autoimmunity but potentially limiting optimal anti-viral responses.

What experimental approaches can distinguish between PELI1's role in T cells versus B cells?

Distinguishing between PELI1's cell-specific functions requires specialized experimental approaches. Mixed bone marrow (BM) chimeras provide a powerful system to assess cell-intrinsic roles. Researchers have generated chimeric mice by reconstituting Rag1−/− mice with mixed BM cells from SJL mice and either wild-type or PELI1-KO mice, allowing simultaneous comparison of wild-type and PELI1-deficient cells in the same environment . Cell-specific knockout models, though not explicitly mentioned in the search results, would be valuable for dissecting PELI1's unique functions in different immune cell types. Adoptive transfer experiments have been effectively used to examine T cell-specific functions of PELI1. For example, transferring wild-type or PELI1-deficient CD4+ T cells into bm12 mice induces lupus-like disease with varying severity, isolating T cell-intrinsic effects . Another approach involves transferring wild-type or PELI1-deficient CD4+ T cells with wild-type B cells into Rag1-KO mice before H1N1 influenza infection, allowing assessment of how T cell-specific PELI1 deficiency affects humoral immunity . In vitro segregation of cell populations followed by specific stimulation and analysis of activation markers can further help delineate cell-specific functions.

What are the key considerations when selecting a PELI1 antibody for specific applications?

When selecting a PELI1 antibody, researchers should consider several critical factors to ensure optimal experimental outcomes. First, application compatibility is essential – verify that the antibody has been validated for your specific application (WB, IP, IHC, or ELISA) with published literature supporting its use . Species reactivity is another crucial consideration; for example, antibody 12053-1-AP has been tested and validated for reactivity with human, mouse, and rat samples . The molecular weight detection range should match your experimental needs – PELI1 is a 418 amino acid protein with a calculated molecular weight of 46 kDa, but is observed between 46-53 kDa in experimental conditions . The antibody's clonality affects its properties; polyclonal antibodies like 12053-1-AP offer high sensitivity but potential batch variation, while monoclonal antibodies provide higher specificity and consistency . Additionally, consider the immunogen used to generate the antibody, as this influences epitope recognition. Finally, examine validation methods used by manufacturers, such as knockout/knockdown controls, to ensure specificity and reliability for your research applications.

What controls should be implemented when using PELI1 antibodies in experimental protocols?

Implementing appropriate controls is essential for validating PELI1 antibody experiments. Positive controls should include samples known to express PELI1, such as THP-1 cells for Western blot or SH-SY5Y cells for immunoprecipitation . Negative controls should utilize PELI1 knockout or knockdown samples when available, which provide the most definitive evidence of antibody specificity. If knockout samples are unavailable, tissues known not to express PELI1 or isotype controls can serve as alternatives. Loading controls are crucial for quantitative Western blot analysis to normalize PELI1 expression levels. For immunohistochemistry applications, include both positive tissue controls (such as mouse brain tissue) and negative controls (primary antibody omission or isotype control antibody) . When optimizing new experimental conditions, performing antibody dilution series (1:50-1:500 for IHC; 1:500-1:1000 for WB) helps determine the optimal signal-to-noise ratio . Additionally, using multiple antibodies targeting different PELI1 epitopes can increase confidence in results, particularly for novel findings. Finally, including recombinant PELI1 protein as a standard can help validate molecular weight and antibody specificity.

How can researchers optimize PELI1 detection in different sample types?

Optimizing PELI1 detection requires tailored approaches for different sample types and detection methods. For protein extraction, choose appropriate lysis buffers based on PELI1's cellular localization – RIPA buffer works well for most applications, but specialized nuclear extraction protocols may improve detection of nuclear PELI1 pools involved in c-Rel regulation . When performing Western blot analysis, transfer conditions should be optimized for PELI1's molecular weight (46-53 kDa); longer transfer times or lower percentage gels may improve detection of higher molecular weight forms resulting from post-translational modifications . For immunohistochemistry, appropriate antigen retrieval is critical – the recommended methods for PELI1 include using TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 . When working with tissue sections, section thickness and fixation protocols should be standardized to ensure reproducible results. For flow cytometry applications (though not explicitly mentioned in search results), permeabilization protocols should be optimized to ensure antibody access to intracellular PELI1. Finally, sample storage conditions can significantly impact PELI1 detection; proteins should be stored with protease inhibitors at appropriate temperatures (-80°C for long-term storage) to prevent degradation.

What experimental systems best demonstrate PELI1's role in T cell activation?

Several experimental systems effectively demonstrate PELI1's role in T cell activation. In vitro T cell stimulation assays comparing wild-type and PELI1-knockout T cells provide direct evidence of PELI1's negative regulatory function. When stimulated with anti-CD3 and anti-CD28 antibodies, PELI1-deficient CD4+ and CD8+ T cells show heightened production of IL-2 and IFN-γ compared to wild-type cells . Proliferation assays using either anti-CD3/CD28 stimulation or PMA plus ionomycin activation demonstrate that PELI1-knockout T cells, particularly CD8+ T cells, display markedly enhanced proliferation capacity . Dose-response experiments are particularly revealing, as PELI1-deficient T cells respond strongly even to low doses of anti-CD3 antibody without CD28 costimulation, while wild-type T cells require both signals for robust activation . To examine PELI1's role in vivo, experimental autoimmune encephalomyelitis (EAE) models using adoptive transfer of wild-type versus PELI1-knockout T cells into Rag1−/− mice demonstrate that PELI1-deficient T cells induce more severe disease with higher frequencies of inflammatory TH17 and TH1 cells .

How can researchers effectively measure PELI1-mediated ubiquitination in experimental settings?

Measuring PELI1-mediated ubiquitination requires specialized approaches to capture this transient post-translational modification. Immunoprecipitation followed by Western blot (IP-WB) is a fundamental technique where the target protein (such as c-Rel or NIK) is immunoprecipitated and then probed with anti-ubiquitin antibodies to detect ubiquitination . To specifically detect K48-linked ubiquitination, which PELI1 mediates for c-Rel degradation, researchers should use antibodies specific for K48-linked polyubiquitin chains . Including proteasome inhibitors (e.g., MG132) in experimental protocols is essential to prevent degradation of ubiquitinated proteins before analysis. In vitro ubiquitination assays using purified components (E1, E2, PELI1 as E3, and substrate) can directly demonstrate PELI1's E3 ligase activity. For cellular systems, expression of tagged ubiquitin (HA-Ub or His-Ub) facilitates detection and purification of ubiquitinated proteins. Proximity ligation assays can visualize PELI1-substrate interactions and ubiquitination events in situ. Finally, mass spectrometry-based approaches allow comprehensive identification of ubiquitination sites and can distinguish between different ubiquitin chain types that may have distinct functional outcomes.

What are the recommended protocols for assessing PELI1 expression in clinical samples?

Assessing PELI1 expression in clinical samples requires careful consideration of sample handling, processing, and detection methods. For protein-level detection in patient samples, Western blot analysis using validated PELI1 antibodies at recommended dilutions (1:500-1:1000) provides quantitative data . Immunohistochemistry offers spatial information about PELI1 expression patterns in tissue sections, with recommended antibody dilutions of 1:50-1:500 and appropriate antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) . Flow cytometry can analyze PELI1 expression in specific immune cell populations from blood or dissociated tissue samples, particularly useful for correlating expression with cell activation states. For high-throughput screening, ELISA-based methods may be developed using validated PELI1 antibodies. At the transcriptional level, quantitative RT-PCR can measure PELI1 mRNA expression using validated primer sets, which is particularly valuable when protein detection is challenging. RNA sequencing provides comprehensive transcriptomic profiles that include PELI1 expression in the context of global gene expression patterns. When analyzing clinical samples, it's crucial to include appropriate controls, standardize collection and processing protocols, and consider potential confounding factors such as medication use, disease stage, and demographic variables.

How do researchers determine if PELI1 is functionally impaired in autoimmune disease models?

Determining functional impairment of PELI1 in autoimmune disease models requires multifaceted approaches. Expression analysis is the first step, examining PELI1 protein and mRNA levels in relevant tissues and cell populations from disease models compared to controls. In human studies, PELI1 levels have been shown to negatively correlate with disease severity in SLE patients . Mutation screening can identify potential genetic alterations affecting PELI1 function, although this wasn't specifically mentioned in the search results. Functional assays comparing PELI1 E3 ubiquitin ligase activity in samples from disease models versus controls directly assess enzymatic function. Substrate ubiquitination analysis specifically examining c-Rel or NIK ubiquitination status can reveal functional defects in PELI1-mediated regulation . Downstream signaling evaluation, particularly noncanonical NF-κB pathway activation, provides indirect evidence of PELI1 dysfunction . Rescue experiments introducing wild-type PELI1 into systems with impaired PELI1 function can confirm causality - overexpression of Peli1 has been shown to inhibit noncanonical NF-κB activation and alleviate lupus-like disease . Finally, therapeutic targeting approaches that enhance PELI1 expression or function in disease models can demonstrate the pathogenic relevance of PELI1 impairment.

What are common challenges in PELI1 Western blot experiments and how can they be resolved?

Western blot experiments with PELI1 antibodies can present several challenges that require specific troubleshooting approaches. Weak signal is a common issue that can be addressed by increasing antibody concentration (within the recommended 1:500-1:1000 range), extending primary antibody incubation time (overnight at 4°C), or using more sensitive detection systems like ECL-Plus . Multiple bands or non-specific binding might occur, which can be minimized by optimizing blocking conditions (5% non-fat milk or BSA), increasing washing stringency, or using lower antibody concentrations. Since PELI1 has an observed molecular weight range of 46-53 kDa, researchers should expect some variation in band size due to post-translational modifications . Inconsistent results between experiments may stem from sample preparation variability; standardizing lysis buffers, protein extraction methods, and including protease inhibitors can improve reproducibility. When comparing PELI1 expression across different cell types or conditions, proper loading controls and quantification methods are essential. If antibody lot-to-lot variation is suspected, maintaining reference samples and performing validation with each new lot is recommended. For particularly difficult samples, enrichment techniques like immunoprecipitation before Western blot analysis may increase detection sensitivity.

How can researchers validate the specificity of PELI1 antibody staining in immunohistochemistry?

Validating PELI1 antibody specificity in immunohistochemistry requires multiple complementary approaches. Knockout/knockdown controls represent the gold standard – comparing staining patterns in PELI1 knockout tissues with wild-type samples provides definitive evidence of specificity . Peptide competition assays, where the immunizing peptide is pre-incubated with the antibody before staining, can confirm epitope-specific binding. Multiple antibody validation involves using different antibodies targeting distinct PELI1 epitopes to confirm consistent staining patterns. Correlation with gene expression data from the same or adjacent tissue sections strengthens confidence in antibody specificity. Isotype controls and primary antibody omission controls help distinguish specific staining from background or non-specific binding. Positive control tissues known to express PELI1 (such as mouse brain tissue) should show the expected staining pattern . Negative control tissues with minimal PELI1 expression should show little to no staining. Antigen retrieval optimization is critical for PELI1 detection; comparison of different methods (TE buffer pH 9.0 versus citrate buffer pH 6.0) helps identify optimal conditions for specific antibody binding . Finally, careful titration of antibody concentration (1:50-1:500) determines the optimal dilution that maximizes specific signal while minimizing background .

What experimental conditions might affect PELI1 protein detection in immune cells?

Multiple experimental conditions can significantly impact PELI1 protein detection in immune cells. Activation state is particularly important, as PELI1 expression is induced upon T cell activation with anti-CD3 and anti-CD28 antibodies . Therefore, the timing of sample collection relative to cell activation influences detection levels. Cell isolation methods can affect protein integrity; mechanical dissociation is often gentler than enzymatic methods that might degrade surface proteins. Fixation and permeabilization protocols are critical for intracellular proteins like PELI1, with different methods potentially exposing different epitopes. Culture conditions, including serum concentration, cell density, and passage number, can alter baseline PELI1 expression in cell lines. For primary immune cells, the presence of cytokines or other stimuli in the culture medium may regulate PELI1 expression. Sample processing time should be minimized, as delayed processing can lead to protein degradation or modification. Protease and phosphatase inhibitors should be included in lysis buffers to preserve post-translational modifications that might affect antibody recognition. Storage conditions affect protein stability; samples should ideally be processed fresh or stored at -80°C with appropriate preservatives. Finally, the subcellular localization of PELI1 may vary depending on cellular context, potentially requiring different extraction methods for comprehensive detection.

When investigating PELI1 in Tfh-mediated immunity, what critical variables need to be controlled?

When investigating PELI1 in Tfh-mediated immunity, several critical variables must be controlled for reproducible and interpretable results. Immunization protocols significantly impact Tfh cell induction; standardizing antigen type (e.g., NP-KLH, NP-OVA), dose (typically 200 μg), route (intraperitoneal or intranasal), and timing of analysis post-immunization is essential . Genetic background effects can influence Tfh responses; experiments should use age and sex-matched mice with appropriate controls, ideally including littermate controls. For chimeric models, irradiation dose (950 rad), bone marrow cell numbers (1 × 107 cells/mouse), and reconstitution period (typically 8 weeks) should be standardized . In adoptive transfer experiments, cell numbers (1 × 106 cells), purity, and activation status of transferred cells affect outcomes . Flow cytometry panels must include comprehensive markers for accurate Tfh identification (typically PD-1+CXCR5+Bcl6+ among CD4+ T cells) and consistent gating strategies across experiments. Germinal center responses should assess both Tfh cells and GC B cells, as PELI1 may affect their interaction . When evaluating humoral immunity, standardized methods for measuring antibody titers (ELISA) and specificity are crucial. In autoimmune models, disease induction protocols (e.g., bm12 transfer model) must be consistent, with careful monitoring of disease progression using established metrics . For viral infection models (such as H1N1), virus strain, dose, route of infection, and methods for assessing viral clearance require standardization .

What are promising therapeutic approaches targeting PELI1 for autoimmune disorders?

Several promising therapeutic approaches targeting PELI1 show potential for autoimmune disorder treatment. PELI1 overexpression strategies have demonstrated efficacy in preclinical models, as overexpression of Peli1 inhibits noncanonical NF-κB activation and alleviates lupus-like disease . Small molecule enhancers of PELI1 E3 ligase activity could boost PELI1's natural inhibitory functions, particularly in settings where PELI1 expression is reduced rather than absent. Targeted protein degradation approaches using PROTACs (Proteolysis Targeting Chimeras) could be designed to enhance PELI1-mediated degradation of specific substrates like NIK or c-Rel. Gene therapy approaches delivering functional PELI1 to specific immune cell populations might correct deficiencies without systemic effects. Cell-specific targeting is crucial since PELI1 has distinct functions in different immune cell types – T cell-specific restoration might prevent autoactive T cell responses, while B cell-specific targeting could reduce autoantibody production . Combination therapies targeting PELI1 alongside established immunosuppressive agents might achieve synergistic effects with reduced toxicity. Biomarker-guided therapy using PELI1 expression levels could help identify patients most likely to benefit from PELI1-targeting approaches, as PELI1 levels negatively correlate with disease severity in SLE patients . Future clinical translation will require careful evaluation of potential side effects, as PELI1 enhancement might impair beneficial immune responses such as anti-viral immunity .

How might single-cell approaches advance our understanding of PELI1 function in diverse immune cell populations?

Single-cell approaches offer transformative potential for understanding PELI1's complex roles across diverse immune cell populations. Single-cell RNA sequencing (scRNA-seq) can reveal cell type-specific expression patterns of PELI1 and correlate these with transcriptional programs and cell states, potentially identifying previously unknown cell populations where PELI1 plays important roles. Single-cell proteomics approaches can measure PELI1 protein expression and post-translational modifications at the individual cell level, capturing heterogeneity missed by bulk analyses. Single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin) can identify regulatory elements controlling PELI1 expression and how these differ between cell types and activation states. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) combines surface protein and transcriptome analysis, allowing correlation of PELI1 expression with immune cell phenotypes. CRISPR-based screens at single-cell resolution can systematically identify genetic interactions with PELI1 in specific immune cell populations. Mass cytometry (CyTOF) with PELI1 antibodies enables high-dimensional phenotyping of PELI1-expressing cells across tissues and disease states. Single-cell spatial transcriptomics can map PELI1 expression within tissue microenvironments, revealing potential interactions with neighboring cells. Finally, longitudinal single-cell profiling during disease progression or therapeutic intervention could identify dynamic changes in PELI1 function and inform optimal timing for therapeutic targeting.

What computational approaches might help predict PELI1 substrates and regulatory networks?

Advanced computational approaches can significantly enhance our understanding of PELI1 substrates and regulatory networks. Structural modeling and molecular docking simulations can predict PELI1-substrate interactions based on protein structures, helping identify novel substrates beyond known targets like c-Rel and NIK . Machine learning algorithms trained on known E3 ligase-substrate pairs can predict additional PELI1 substrates based on sequence features, structural properties, and interaction patterns. Network analysis of protein-protein interaction data can place PELI1 within broader signaling networks, revealing potential cross-talk with other pathways. Transcriptomic data integration comparing wild-type and PELI1-deficient cells under various stimulation conditions can identify genes and pathways indirectly regulated by PELI1. Pathway enrichment analysis of differentially expressed genes in PELI1-deficient models can highlight biological processes most affected by PELI1 dysfunction. UbiSite and similar tools can predict potential ubiquitination sites on candidate PELI1 substrates, guiding experimental validation. Systems biology approaches integrating multi-omics data (transcriptomics, proteomics, ubiquitinomics) can construct comprehensive PELI1 regulatory networks across different cell types and conditions. Temporal modeling of signaling dynamics can predict how PELI1-mediated regulation affects the kinetics of immune responses. Finally, CRISPR screen data analysis can identify synthetic lethal interactions with PELI1, revealing potential therapeutic targets for contexts where PELI1 is dysfunctional.