PELI3 Antibody

What is PELI3 and why is it important in cellular signaling research?

PELI3 (Pellino E3 ubiquitin protein ligase family member 3) is a critical component of various inflammatory signaling pathways, including Toll-like receptor signaling, NOD2 signaling, and TNF signaling pathways. The Pellino3 protein, encoded by the PELI3 gene, contains a RING domain with E3 ligase activity that facilitates the activation of mitogen-activated protein kinases (MAPKs) such as JNK and p38 . The human version of Pellino 3 has a canonical amino acid length of 469 residues and a protein mass of 50.8 kilodaltons, with four distinct isoforms identified to date . Pellino3's role in ubiquitination processes and cell death regulation makes it a significant target for researchers investigating inflammatory diseases, liver injuries, and cellular stress responses.

How do different isoforms of Pellino 3 affect antibody selection for experimental procedures?

When selecting antibodies for Pellino 3 detection, researchers must consider the presence of multiple isoforms (at least four have been identified in humans). These isoforms may display differential expression patterns across tissues and under various physiological conditions. For optimal experimental outcomes, researchers should:

• Determine which isoform(s) are relevant to their specific research question

• Select antibodies that either recognize conserved regions (for pan-Pellino 3 detection) or isoform-specific epitopes

• Validate antibody specificity using positive controls expressing the target isoform(s)

• Consider using recombinant expression constructs such as Flag-hPellino3a, Flag-hPellino3b, 6xMyc-hPellino3a, and 6xMyc-hPellino3b when investigating isoform-specific functions

The choice between these approaches depends on whether the research aims to study general Pellino 3 functions or isoform-specific roles in particular cellular contexts.

What are the optimal conditions for Western blot detection of PELI3 in different tissue samples?

For optimal Western blot detection of PELI3 across different tissue samples, researchers should implement the following protocol:

• Sample preparation:

  • For liver tissue: Homogenize in RIPA buffer containing protease inhibitors and phosphatase inhibitors

  • For cultured cells: Lyse directly in 1X SDS sample buffer or extract using NP-40 lysis buffer

• Protein separation:

  • Use 10-12% SDS-PAGE gels for optimal resolution of the ~50.8 kDa Pellino 3 protein

  • Load 25-50 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody dilution: 1:500 to 1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Secondary antibody: HRP-conjugated anti-mouse or anti-rabbit (depending on primary antibody species) at 1:5000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Expose to film or use digital imaging systems with exposure times of 30 seconds to 5 minutes

Different tissue types may require optimization of lysis buffers and protein extraction methods to ensure consistent detection of Pellino 3 .

How can researchers effectively perform immunoprecipitation of PELI3 for protein interaction studies?

For effective immunoprecipitation (IP) of PELI3 to study protein interactions, researchers should follow this methodological approach:

• Cell/tissue preparation:

  • Harvest cells or tissue and lyse in a non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemented with protease and phosphatase inhibitors

  • Clarify lysates by centrifugation at 14,000g for 15 minutes at 4°C

• Pre-clearing (to reduce non-specific binding):

  • Incubate lysates with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of Pellino 3 antibody (such as Pellino 3 (B-3) Antibody) to 500-1000 μg of pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Alternatively, use pre-coupled agarose conjugated antibodies like Pellino 3 (B-3) AC Antibody for direct pull-down

• Bead capture and washing:

  • Add 40 μl of Protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with lysis buffer

  • For studying ubiquitination, include 2% SDS in the first wash buffer followed by dilution to 0.2% SDS for subsequent washes

• Elution and analysis:

  • Elute bound proteins by boiling in 2X SDS sample buffer for 5 minutes

  • Analyze by Western blot using antibodies against PELI3 and potential interacting partners

For studying specific interactions, such as between Pellino3 and GSK3β or JNK1/2, this method can be effectively coupled with subsequent Western blot analysis .

How should researchers design experiments to investigate PELI3's role in acetaminophen-induced liver injury?

When investigating PELI3's role in acetaminophen (APAP)-induced liver injury, researchers should implement a comprehensive experimental design that addresses multiple aspects of the injury mechanism:

• Animal model setup:

  • Use Peli3−/− knockout (KO) mice and wild-type (WT) controls (C57BL/6 background)

  • Consider adenovirus-mediated Peli3 knockdown (KD) mice as an alternative model

  • Age- and sex-match all experimental groups (typically 8-12 weeks old)

  • Administer APAP (300-500 mg/kg) via intraperitoneal injection after overnight fasting

• Assessment parameters:

  • Monitor survival rates (if using lethal APAP doses)

  • Collect serum at 6, 12, and 24 hours post-APAP for ALT/AST measurement

  • Harvest liver tissue for histological analysis (H&E staining)

  • Quantify immune cell infiltration using flow cytometry

  • Measure mitochondrial damage markers and ROS levels

• Mechanistic investigations:

  • Isolate primary hepatocytes from WT and Peli3−/− mice for in vitro studies

  • Assess mitochondrial translocation of GSK3β via subcellular fractionation

  • Examine JNK phosphorylation levels by Western blotting

  • Perform ubiquitination assays to detect K63-mediated polyubiquitination of GSK3β

  • Conduct rescue experiments using adenoviral expression of wild-type Pellino3 versus catalytically inactive mutants

• Controls and validation:

  • Include vehicle-treated groups for both genotypes

  • Confirm genotypes by PCR using appropriate primers

  • Validate Peli3 deletion by qRT-PCR

  • Test for compensatory expression of other Pellino family members

This experimental design allows for comprehensive characterization of PELI3's role in APAP hepatotoxicity through multiple complementary approaches .

What are the key considerations for designing cellular assays to study PELI3's E3 ligase activity?

To effectively study PELI3's E3 ligase activity in cellular assays, researchers should consider the following key experimental design elements:

• Expression system selection:

  • Choose appropriate cell lines that either naturally express PELI3 (primary hepatocytes) or are easily transfectable (HEK293 cells)

  • Consider using cell lines relevant to the biological context being studied (e.g., immune cells for inflammatory pathway studies)

• Construct design:

  • Generate wild-type and catalytically inactive PELI3 mutants (mutations in the RING domain)

  • Include appropriate epitope tags (Flag, Myc, HA) for detection and immunoprecipitation

  • Design constructs for both human isoforms (Pellino3a and Pellino3b) and mouse Pellino3

• Ubiquitination assay setup:

  • Co-express PELI3 with potential substrates (e.g., GSK3β, JNK1, JNK2)

  • Include HA-tagged ubiquitin constructs (wild-type and K63-only variants)

  • Perform ubiquitination assays under denaturing conditions to eliminate non-covalent interactions

  • Use proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

• Controls and validation:

  • Include vector-only controls

  • Use siRNA/shRNA-mediated knockdown of endogenous PELI3

  • Compare wild-type PELI3 with catalytically inactive mutants

  • Validate physical interactions using co-immunoprecipitation before ubiquitination studies

• Detection methods:

  • Use immunoblotting with specific antibodies against ubiquitin, K63-linkage, and target proteins

  • Employ size-shift analysis to detect multiple ubiquitination events

  • Consider mass spectrometry to identify specific ubiquitination sites on target proteins

This methodological framework provides a comprehensive approach to characterizing PELI3's E3 ligase activity and substrate specificity in cellular contexts .

How can researchers address inconsistent PELI3 detection in immunofluorescence studies?

When encountering inconsistent Pellino 3 detection in immunofluorescence studies, researchers should systematically troubleshoot using the following approach:

• Fixation optimization:

  • Test multiple fixation methods:

    • 4% paraformaldehyde (10-15 minutes at room temperature)

    • Methanol (-20°C for 10 minutes)

    • Acetone (-20°C for 5 minutes)

    • Combination fixation (PFA followed by methanol permeabilization)

  • Determine optimal fixation based on epitope accessibility and cellular compartment of interest

• Permeabilization refinement:

  • Compare different permeabilization agents:

    • 0.1-0.5% Triton X-100

    • 0.1-0.5% Saponin

    • 0.1% SDS (for more stringent permeabilization)

  • Adjust permeabilization time (5-15 minutes) based on cell type and thickness

• Blocking and antibody parameters:

  • Use 5-10% serum from the species of secondary antibody

  • Add 1% BSA to reduce non-specific binding

  • Test different antibody dilutions (1:50 to 1:500)

  • Extend primary antibody incubation (overnight at 4°C rather than 1-2 hours)

  • Consider using FITC-conjugated anti-Pellino 3 antibodies for direct detection

• Signal amplification and detection:

  • Implement tyramide signal amplification for low-abundance proteins

  • Use high-sensitivity detection systems

  • Optimize exposure settings on microscopes

  • Consider spectral unmixing for multi-label experiments

• Positive controls and validation:

  • Include cells transfected with tagged PELI3 constructs as positive controls

  • Validate antibody specificity using PELI3 knockout or knockdown samples

  • Compare results with cells known to express high levels of PELI3

Implementation of this systematic troubleshooting approach can significantly improve consistency and specificity in Pellino 3 immunofluorescence detection .

What strategies should be employed when Western blot results for PELI3 conflict with qRT-PCR data?

When Western blot results for PELI3 protein expression conflict with qRT-PCR data measuring PELI3 mRNA levels, researchers should implement the following investigative strategy:

• Methodological validation:

  • Confirm primer specificity for qRT-PCR (test against known positive and negative controls)

  • Verify antibody specificity using PELI3 knockout samples or blocking peptides

  • Re-sequence PCR products to confirm target amplification

  • Test multiple validated antibodies targeting different PELI3 epitopes

• Post-transcriptional regulation assessment:

  • Measure PELI3 mRNA stability using actinomycin D chase experiments

  • Investigate microRNA regulation using prediction algorithms and validation assays

  • Examine alternative splicing with isoform-specific primers

  • Consider nonsense-mediated decay of specific transcripts

• Post-translational modification and protein stability analysis:

  • Treat samples with proteasome inhibitors (MG132) to assess protein degradation rates

  • Examine ubiquitination patterns of PELI3 itself

  • Test various protein extraction methods to ensure complete solubilization

  • Use phosphatase inhibitors to preserve potential phosphorylated forms

• Experimental design considerations:

  • Implement time-course studies to capture temporal discrepancies between mRNA and protein

  • Include tissue-specific positive controls known to express PELI3

  • Normalize to multiple housekeeping genes/proteins

  • Consider subcellular fractionation (PELI3 may relocalize under certain conditions)

• Technical approach to resolve discrepancies:

  Observation	Possible Explanation	Recommended Investigation
  High mRNA, Low protein	Rapid protein degradation	Proteasome inhibitor treatment
  Low mRNA, High protein	Protein stability	Cycloheximide chase assay
  Variable results between experiments	Protocol inconsistency	Standardize lysate preparation
  Discrepancies in specific tissues	Tissue-specific regulation	Compare multiple tissue sources
  Multiple bands on Western blot	Isoforms or degradation	Isoform-specific antibodies

This comprehensive approach helps determine whether discrepancies represent biological phenomena or technical artifacts, enabling more accurate interpretation of PELI3 expression data .

How can PELI3 knockout models inform therapeutic strategies for acetaminophen-induced liver injury?

PELI3 knockout models provide significant insights that can inform therapeutic strategies for acetaminophen (APAP)-induced liver injury through several mechanistic pathways:

• GSK3β pathway modulation:

  • Peli3−/− knockout mice show reduced phosphorylation at serine 9 and decreased mitochondrial translocation of GSK3β

  • This reduction correlates with decreased JNK phosphorylation and mitochondrial translocation

  • Therapeutic strategies targeting GSK3β phosphorylation or mitochondrial translocation could mimic the protective effects seen in PELI3-deficient models

• Mitochondrial protection mechanisms:

  • Primary hepatocytes from Peli3−/− mice demonstrate decreased mitochondrial damage

  • Reduced mitochondrial reactive oxygen species (ROS) levels are observed

  • Lysosomal damage is also attenuated in these models

  • Therapeutic approaches focusing on mitochondrial preservation during APAP toxicity would align with PELI3 knockout protective mechanisms

• Inflammation modulation:

  • PELI3 knockout mice exhibit reduced immune cell infiltration upon APAP treatment

  • Inflammatory biomarkers are decreased compared to wild-type mice

  • Anti-inflammatory interventions targeting pathways downstream of PELI3 could provide hepatoprotection

• Translational applications:

  • Development of small molecule inhibitors specifically targeting PELI3's E3 ligase activity

  • Design of peptide-based inhibitors that disrupt PELI3-GSK3β interaction

  • Creation of liver-targeted siRNA or antisense oligonucleotides to reduce PELI3 expression during acute toxicity

  • Screening for compounds that inhibit K63-mediated polyubiquitination of GSK3β

These findings suggest that therapeutic strategies targeting the PELI3-GSK3β-JNK axis could provide effective protection against APAP-induced liver injury, particularly when administered in the early phases of toxicity development .

What are the challenges and opportunities in developing PELI3-targeting therapeutic strategies for inflammatory conditions?

The development of PELI3-targeting therapeutic strategies for inflammatory conditions presents several challenges and opportunities:

Challenges:

• Functional redundancy:

  • Pellino family members (Pellino1, Pellino2, and Pellino3) share structural similarities

  • Potential compensatory mechanisms may limit efficacy of PELI3-specific inhibition

  • Distinguishing unique functions of PELI3 from other family members is crucial

• Pathway complexity:

  • PELI3 is involved in diverse inflammatory signaling pathways (Toll-like receptor, NOD2, and TNF signaling)

  • Different isoforms may have distinct or even opposing functions in specific contexts

  • Temporal dynamics of PELI3 activity during inflammatory responses are not fully characterized

• Target specificity:

  • Developing inhibitors specific to PELI3's E3 ligase activity without affecting other RING domain-containing proteins

  • Ensuring tissue-specific delivery to minimize off-target effects

  • Maintaining selectivity for pathological versus physiological PELI3 functions

Opportunities:

• Novel mechanistic insights:

  • PELI3's role in GSK3β ubiquitination and mitochondrial translocation provides a unique therapeutic target

  • K63-specific ubiquitination activity offers potential for highly selective intervention

  • The demonstrated protective effect of PELI3 ablation in liver injury models suggests therapeutic potential

• Emerging technological approaches:

  • PROTAC (Proteolysis Targeting Chimera) technology could be applied to selectively degrade PELI3

  • Structure-based drug design targeting the RING domain or substrate interaction surfaces

  • Antisense oligonucleotides for isoform-specific modulation of PELI3 expression

• Therapeutic applications:

  • Acute liver injury (particularly acetaminophen overdose)

  • Inflammatory bowel disease (based on PELI3's role in NOD2 signaling)

  • Autoimmune conditions where excessive MAPK activation contributes to pathology

  • Potential application in cancer contexts where apoptosis regulation is disrupted

• Biomarker development:

  • PELI3 expression or activity patterns could serve as predictive biomarkers for treatment response

  • Monitoring PELI3-mediated ubiquitination as a pharmacodynamic marker

How do different anti-PELI3 antibodies compare in terms of specificity and sensitivity across applications?

A comprehensive comparison of anti-PELI3 antibodies reveals significant variations in performance across different experimental applications:

Key considerations for antibody selection based on application:

• Western blot applications:

  • Pellino 3 (B-3) Antibody offers superior sensitivity and consistent performance

  • Polyclonal antibodies may detect multiple bands due to post-translational modifications

  • Pre-adsorbed antibodies reduce background in complex tissue lysates

• Immunofluorescence studies:

  • FITC-conjugated antibodies eliminate secondary antibody cross-reactivity issues

  • Pellino 3 (B-3) Antibody provides excellent cellular localization information

  • Fixation method significantly impacts epitope accessibility with certain antibodies

• Immunoprecipitation experiments:

  • Agarose-conjugated antibodies (Pellino 3 (B-3) AC) offer superior pull-down efficiency

  • Non-conjugated antibodies require optimization of antibody-to-bead ratios

  • Species matching between primary antibody and IP antibody improves detection in subsequent Western blots

• Cross-species applications:

  • Few antibodies recognize PELI3 across human, mouse, and rat samples

  • Species-specific antibodies may be necessary for specialized applications

  • Sequence alignment confirms epitope conservation when using cross-reactive antibodies

This comparative analysis enables researchers to select the optimal PELI3 antibody based on their specific experimental requirements, tissue source, and application needs .

How should researchers design PELI3 studies to investigate its role in different inflammatory signaling pathways?

To effectively investigate PELI3's role in different inflammatory signaling pathways, researchers should implement a multi-faceted experimental design strategy:

• Pathway-specific stimulation protocols:

  Signaling Pathway	Recommended Stimuli	Optimal Duration	Key Readouts	PELI3 Role Assessment
  TLR Signaling	LPS (TLR4), Pam3CSK4 (TLR1/2), CpG DNA (TLR9)	0-24 hours	NF-κB activation, MAPK phosphorylation	E3 ligase activity toward TRAF molecules
  NOD2 Signaling	MDP (NOD2 ligand)	0-12 hours	RIP2 ubiquitination, inflammatory cytokines	Negative regulation of NOD2-induced cytokines
  TNF Signaling	TNF-α	0-8 hours	Cell viability, caspase activation	Regulation of apoptotic responses

• Cell type considerations:

  • Primary immune cells (macrophages, dendritic cells) for physiological relevance

  • Hepatocytes for investigating APAP-induced liver injury mechanisms

  • Cell lines for mechanistic studies (RAW264.7, HEK293T with pathway reporters)

  • Tissue-specific conditional knockout models to address cell-type specific functions

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout of PELI3

  • siRNA/shRNA knockdown for acute depletion studies

  • Rescue experiments with wild-type vs. catalytically inactive PELI3

  • Isoform-specific manipulation using targeted constructs

  • Domain mutants to dissect functional regions

• Ubiquitination analysis methodology:

  • Immunoprecipitation under denaturing conditions

  • Ubiquitin linkage-specific antibodies (K48 vs. K63)

  • Mass spectrometry to identify ubiquitination sites

  • In vitro ubiquitination assays with purified components

  • Proteasome inhibitors to distinguish degradative vs. regulatory ubiquitination

• Temporal dynamics assessment:

  • Time-course experiments capturing early and late events

  • Pulse-chase studies for protein turnover analysis

  • Live-cell imaging with fluorescently tagged constructs

  • Sequential chromatin immunoprecipitation for transcriptional effects

This comprehensive experimental approach enables researchers to dissect PELI3's specific roles across different inflammatory signaling cascades, distinguishing its functions from other Pellino family members and identifying pathway-specific mechanisms .