rnf146 Antibody

What is RNF146 and why is it important in cellular signaling?

RNF146 is an E3 ubiquitin ligase that binds to poly-ADP-ribosylated (PARsylated) proteins and mediates their ubiquitination and subsequent degradation. It contains both RING and WWE domains that are crucial for its function . The protein plays a significant role in various cellular processes, most notably as an activator of the Wnt signaling pathway by mediating the ubiquitination of PARsylated AXIN1 and AXIN2, which are key components of the beta-catenin destruction complex . RNF146 acts in cooperation with tankyrase proteins (TNKS and TNKS2), which mediate PARsylation of target proteins . Beyond Wnt signaling, RNF146 serves as a neuroprotective protein that prevents nuclear translocation of AIFM1 in a PAR-binding dependent manner and protects against cell death induced by DNA damaging agents .

What are the key structural domains of RNF146 that antibodies typically target?

RNF146 contains two critical functional domains that researchers often target with antibodies:

• The RING domain: Essential for E3 ligase activity, this domain is responsible for the protein's ability to catalyze ubiquitination reactions .

• The WWE domain: Critical for recognition and binding of PARsylated proteins, this domain allows RNF146 to specifically identify its substrates .

When selecting antibodies for RNF146 detection, researchers should consider which domain is most relevant to their research question. For instance, studies focused on substrate recognition might benefit from antibodies targeting the WWE domain, while investigations of ubiquitination activity might prioritize RING domain-specific antibodies. The N-terminal region (residues 1-200) is also commonly used as an immunogen for antibody production .

How can I verify the specificity of an RNF146 antibody in my experimental system?

To verify antibody specificity for RNF146, implement the following methodological approach:

• Positive and negative controls: Use cell lines with known RNF146 expression levels. RNF146 knockout (KO) cell lines generated using CRISPR/Cas9 technology serve as excellent negative controls .

• Protein size verification: Confirm that your antibody detects a protein of the expected molecular weight (approximately 33-39 kDa) .

• Knockdown validation: Perform siRNA-mediated knockdown of RNF146 and verify reduced signal intensity in Western blots compared to control siRNA (e.g., luciferase siRNA) .

• Rescue experiments: Re-express siRNA-resistant RNF146 variants in knockdown cells to restore the signal, confirming antibody specificity .

• Cross-reactivity assessment: If working with non-human samples, verify species cross-reactivity, as RNF146 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

How can RNF146 antibodies be optimized for studying the interaction between RNF146 and tankyrase proteins?

Studying RNF146-tankyrase interactions requires specialized antibody application strategies:

• Co-immunoprecipitation optimization: When performing co-IP experiments to study RNF146-tankyrase interactions, consider that wild-type RNF146 can degrade itself, resulting in extremely low expression levels compared to mutant forms . Use RNF146 ΔRING mutants for co-IP studies, as they express at higher levels while maintaining tankyrase binding capacity .

• Domain-specific antibodies: Select antibodies that specifically recognize regions outside the tankyrase interaction motifs. RNF146 contains five potential tankyrase-binding motifs (I-V) with motif I (residues 193-199) being the most conserved . The C-terminal region of RNF146 is crucial for TNKS binding, as truncations (RNF146(1-183)) disrupt this interaction .

• Subcellular localization studies: Use immunofluorescence with confocal microscopy to visualize RNF146 and tankyrase co-localization. This approach can reveal how RNF146 regulates TNKS and TNKS2 subcellular localization, potentially preventing aggregation at centrosomal locations .

• Cross-linking approaches: Consider implementing chemical cross-linking prior to immunoprecipitation to stabilize transient interactions between RNF146 and tankyrases for more robust detection.

What methods can I use to study RNF146's role in the β-glucan-induced immune training state?

To investigate RNF146's involvement in β-glucan-induced immune training:

• Expression analysis during immune training: Temporally monitor RNF146 expression levels in models like RAW264.7 macrophages treated with β-glucan (100 ng/mL) for 24 hours, followed by a resting period and subsequent LPS challenge .

• Immunofluorescence co-localization: Perform dual immunofluorescence staining of RNF146 and Akt in trained versus untrained cells to evaluate their spatial relationship during immune training states .

• Pathway inhibition: Use RNF146 knockdown/knockout models to determine if the protective effects of β-glucan pretreatment are dependent on RNF146 expression.

• Protein-protein interaction mapping: Apply co-immunoprecipitation techniques to identify changes in RNF146's interaction network during the different phases of immune training (priming, resting, and challenge).

• Cytokine profile analysis: Combine RNF146 antibody-based detection methods with ELISA quantification of inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-10) to correlate RNF146 expression with functional immune outputs .

Research has shown that RNF146 and Akt are downregulated during the immunosuppression period of sepsis but increased after β-glucan pretreatment, which induces trained immunity in septic mice .

How can I use RNF146 antibodies to identify novel substrates of this E3 ligase?

To identify novel RNF146 substrates, implement the following methodological approach:

• Proteome-wide analysis: Perform quantitative proteomics comparing wild-type cells with RNF146 knockout cells to identify proteins that accumulate in the absence of RNF146 . This approach has successfully identified OTUD5, PARP10, and SARDH as substrates of RNF146 .

• Validation strategy:

  • Western blot confirmation: Verify protein level changes for candidate substrates in RNF146 KO cells versus wild-type cells

  • Reconstitution experiments: Re-express wild-type RNF146 or domain deletion mutants (ΔWWE or ΔRING) in RNF146 KO cells to confirm rescue of substrate degradation

  • Domain dependency analysis: Determine if substrate recognition depends on the WWE domain (PAR-binding) or requires additional interactions

• Substrate interaction analysis: Perform co-immunoprecipitation experiments with RNF146 ΔRING mutants (to prevent substrate degradation) to confirm direct interaction with candidate substrates .

• Tankyrase dependency: Compare proteomic data from RNF146 KO cells with TNKS1/2 double knockout cells to distinguish between tankyrase-dependent and tankyrase-independent RNF146 substrates .

• PARylation inhibition: Use PARP inhibitors (e.g., olaparib) or tankyrase inhibitors (e.g., XAV939) to determine if substrate recognition is dependent on PARylation .

What are the optimal conditions for using RNF146 antibodies in different experimental techniques?

Different experimental techniques require specific optimization of RNF146 antibody use:

• Western Blotting:

  • Sample preparation: Use RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail

  • Dilution ratios: Start with manufacturer-recommended dilutions (typically 1:1000) and optimize as needed

  • Blocking: Use 5% non-fat dry milk or BSA in TBST

  • Detection: Both chemiluminescence and fluorescence-based detection systems are suitable

• Immunofluorescence:

  • Fixation: 4% paraformaldehyde in PBS

  • Permeabilization: 0.1% Triton X-100

  • Blocking: 2% bovine serum albumin (BSA)

  • Primary antibody incubation: Overnight at 4°C

  • Secondary antibody: Fluorophore-conjugated antibodies, 1 hour at room temperature

  • Imaging: Confocal microscopy (e.g., LSM 710, Zeiss)

• Immunoprecipitation:

  • For RNF146 self-degradation studies, consider using ΔRING mutants to achieve higher expression levels

  • Include protease inhibitors and phosphatase inhibitors in lysis buffers

  • Consider mild detergents for preserving protein-protein interactions

• Immunohistochemistry:

  • RNF146 antibodies suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) are available

  • Follow standard antigen retrieval protocols for formalin-fixed tissues

How should I design experiments to study the allosteric activation of RNF146 by poly(ADP-ribose)?

To study the allosteric activation of RNF146 by poly(ADP-ribose):

• Domain-specific antibody selection: Choose antibodies that can detect conformational changes in the RNF146 RING-WWE domains upon PAR binding .

• Functional assays:

  • In vitro ubiquitination assays: Compare ubiquitination activity of purified RNF146 with and without poly(ADP-ribose) using substrates like AXIN1

  • TNKS/TNKS2 co-immunoprecipitation: Evaluate how PAR binding affects RNF146's interaction with tankyrase proteins

  • Domain mutation analysis: Create point mutations in the WWE domain and assess their impact on PAR binding and RNF146 activation

• PAR modulation approaches:

  • PARP inhibitors: Use inhibitors like olaparib to decrease cellular PARylation and observe effects on RNF146 activity

  • Tankyrase inhibitors: Apply XAV939 to specifically inhibit tankyrase-mediated PARylation and determine effects on RNF146 substrate levels

  • PAR-binding mutants: Compare the activity of wild-type RNF146 with WWE domain mutants defective in PAR binding

• Subcellular localization: Track changes in RNF146 localization upon PAR generation using immunofluorescence with confocal microscopy .

• Structural considerations: RNF146(RING-WWE)/UbcH5a/iso-ADPr complex crystallization has been achieved, revealing important structural features of activated RNF146 .

What controls should be included when using RNF146 antibodies in knockout/knockdown validation experiments?

When validating RNF146 antibodies in knockout/knockdown experiments, include these essential controls:

• Genetic controls:

  • Negative control: RNF146 knockout cell lines generated using CRISPR/Cas9 gene editing technology

  • Positive control: Wild-type cells with known RNF146 expression

  • siRNA controls: Use control siRNAs targeting non-related genes (e.g., luciferase) alongside RNF146-specific siRNAs

  • Rescue controls: Re-express siRNA-resistant RNF146 constructs to restore function and antibody detection

• Domain-specific controls:

  • Domain deletion mutants: Express RNF146 ΔWWE or ΔRING deletion mutants to validate domain-specific antibodies

  • Point mutants: Use functionally relevant mutants (e.g., G199V in motif I or G337V/G338V in motif IV) that affect specific interactions

• Experimental technique controls:

  • Loading controls: Include detection of housekeeping proteins (e.g., tubulin) in Western blots

  • Secondary antibody-only controls: Verify absence of non-specific binding

  • Cross-reactivity controls: If applicable, test antibodies on tissue/cells from other species to confirm specificity

• Functional validation:

  • Substrate accumulation: Confirm accumulation of known RNF146 substrates (e.g., AXIN1, BLZF1, AMOT) in knockout/knockdown conditions

  • Pathway activation: Monitor Wnt pathway activity changes (e.g., β-catenin levels) in the absence of RNF146

How do I address inconsistent RNF146 antibody detection in different cell types or tissues?

When facing inconsistent RNF146 detection across different samples:

• Expression level considerations:

  • RNF146 is ubiquitously expressed across many tissue types, but expression levels may vary

  • Self-degradation may lead to lower steady-state levels of wild-type RNF146 compared to catalytically inactive mutants

• Sample preparation optimization:

  • Adjust lysis buffer composition: For cytoplasmic and nuclear fractions, use RIPA buffer with protease inhibitors

  • Consider protein extraction protocols specific to different tissue types

  • Optimize protein loading concentrations based on expected RNF146 abundance

• Antibody selection strategy:

  • Test multiple antibodies targeting different epitopes of RNF146

  • For tissues with potential splice variants, select antibodies recognizing conserved regions

  • Consider RNF146 can exist in up to 2 different isoforms in humans

• Validation approaches:

  • Perform mRNA expression analysis alongside protein detection

  • Use recombinant RNF146 as a positive control in challenging samples

  • Implement immunoprecipitation followed by Western blot for enrichment when dealing with low abundance

• Experimental conditions:

  • Adjust exposure times for Western blots based on expected abundance

  • For immunofluorescence, optimize antibody concentration and incubation times for each cell type

How can I distinguish between RNF146-dependent and tankyrase-dependent effects in my research?

To differentiate between RNF146 and tankyrase-dependent effects:

• Genetic approach:

  • Compare phenotypes between RNF146 knockout cells, TNKS1/2 double knockout cells, and triple knockout models

  • Create rescue cell lines expressing RNF146 mutants defective in tankyrase binding (motif I G199V mutation)

• Pharmacological approach:

  • Use tankyrase-specific inhibitors (e.g., XAV939) in both wild-type and RNF146 knockout backgrounds

  • Compare effects of PARP inhibitors (e.g., olaparib) that inhibit multiple PARPs versus tankyrase-specific inhibitors

• Substrate classification:

  • Identify tankyrase-dependent RNF146 substrates (e.g., SARDH) versus tankyrase-independent substrates (e.g., PARP10)

  • Analyze substrate accumulation patterns across different knockout models

• Interaction domain analysis:

  • Examine how mutations in RNF146 tankyrase-binding motifs affect substrate recognition

  • Use RNF146 constructs lacking the WWE domain to identify PAR-independent functions

• Quantitative proteomics:

  • Compare proteome changes in RNF146KO versus TNKS1/2DKO cells to systematically classify proteins as primarily regulated by either E3 ligase activity or PARylation

What approaches can resolve contradictory results when studying RNF146 in different experimental models?

When faced with contradictory results across experimental models:

• Model system differences analysis:

  • Compare RNF146 expression levels and isoform distribution across your experimental models

  • Evaluate the expression status of key RNF146 interactors (TNKS, TNKS2, PARP1) in each model

  • Consider cell line authentication to verify genetic backgrounds

• Pathway context assessment:

  • Analyze baseline Wnt pathway activation status across models

  • Evaluate DNA damage response pathway activity, which may affect RNF146 function

  • Consider the influence of cellular stress levels on RNF146-dependent processes

• Methodological standardization:

  • Implement identical antibody-based detection protocols across all models

  • Standardize cell culture conditions, passage numbers, and confluency

  • Use consistent lysis and protein extraction protocols

• Multi-level validation approach:

  • Combine antibody-based protein detection with mRNA expression analysis

  • Validate key findings using orthogonal techniques (e.g., mass spectrometry)

  • Implement both genetic (CRISPR, siRNA) and pharmacological (XAV939, olaparib) approaches

• Functional readouts:

  • Move beyond RNF146 detection to examine downstream effects on known substrates

  • Quantify functional outcomes like Wnt pathway activation or cell survival after DNA damage

  • Develop model-specific positive controls that verify antibody functionality in each system

How can RNF146 antibodies be integrated into high-throughput screening approaches?

For high-throughput screening applications with RNF146 antibodies:

• Automated immunofluorescence platforms:

  • Develop RNF146 antibody-based high-content imaging assays to screen for compounds affecting RNF146 localization or expression

  • Multiplex with antibodies against known substrates to monitor degradation dynamics

• ELISA-based screening:

  • Design sandwich ELISA systems using capture and detection antibodies against different RNF146 epitopes

  • Develop competitive ELISAs to screen for compounds that disrupt RNF146-substrate interactions

• Protein array applications:

  • Use purified RNF146 antibodies to probe protein arrays for novel interactors

  • Create reverse-phase protein arrays with tumor samples to correlate RNF146 expression with clinical outcomes

• Genome-wide approaches:

  • Combine RNF146 antibody-based detection with CRISPR screens to identify genes affecting RNF146 stability or activity

  • Integrate with proteomics to identify factors that modulate the RNF146 substrate landscape

• Drug discovery platforms:

  • Develop cellular assays using RNF146 antibodies to screen for compounds that stabilize or destabilize RNF146

  • Create reporter systems combining RNF146 antibody-based detection with substrate readouts

What emerging technologies might enhance our understanding of RNF146's role in cellular signaling networks?

Emerging technologies for studying RNF146 in signaling networks include:

• Proximity labeling proteomics:

  • Engineer BioID or APEX2 fusion constructs with RNF146 to identify proximal proteins in living cells

  • Compare the RNF146 proximitome in normal versus stressed conditions

• Live-cell imaging:

  • Develop RNF146-specific nanobodies compatible with live-cell applications

  • Combine with fluorescent ubiquitin sensors to visualize RNF146-dependent ubiquitination in real-time

• Single-cell analysis:

  • Implement RNF146 antibodies in mass cytometry (CyTOF) panels to correlate RNF146 expression with cellular states

  • Combine with single-cell RNA-seq to link transcriptional signatures with RNF146 protein levels

• Structural biology approaches:

  • Use conformation-specific antibodies to probe RNF146 structural changes upon PAR binding

  • Apply cryo-EM to study RNF146 in complex with its substrates and E2 enzymes

• In vivo applications:

  • Develop RNF146 antibodies suitable for intravital imaging

  • Create RNF146 reporter mice with endogenous tagging for longitudinal studies