PDZRN3 Antibody

What is PDZRN3 and what cellular functions does it regulate?

PDZRN3 is an E3 ubiquitin-protein ligase with a molecular weight of 119.6 kDa in humans, consisting of 1066 amino acid residues in its canonical form. The protein contains PDZ domains and a RING finger domain, with subcellular localization primarily in the cytoplasm . PDZRN3 functions in protein ubiquitination processes and plays crucial roles in several cellular mechanisms:

• Regulation of cell proliferation and suppression of apoptosis in myoblasts through maintenance of cyclin A2 expression

• Modulation of Wnt signaling pathways, particularly by attenuating the Wnt/β-catenin canonical pathway while promoting the Wnt/PCP (Planar Cell Polarity) pathway

• Contribution to vascular development and angiogenic processes

• Influence on DNA damage repair mechanisms via regulation of Mre11 expression

Three different isoforms of the protein have been reported, with varying tissue expression patterns.

What is the tissue distribution pattern of PDZRN3?

PDZRN3 demonstrates a broad tissue expression profile with significant variability in expression levels:

• High expression: Heart, skeletal muscle, and liver

• Moderate to low expression: Brain, colon, small intestine, placenta, and lung

• Developmental expression: Present in endothelial cells during embryonic development, particularly observable in E12.5 yolk sac and E14.5 brain

• Postnatal expression: Detected in retinal and aortic endothelial cells at day 7 (P7)

This expression pattern suggests tissue-specific regulatory roles, particularly in muscle development and vascular formation.

What experimental applications are PDZRN3 antibodies commonly used for?

PDZRN3 antibodies are employed in multiple research techniques:

• Western Blot (WB): For detection and quantification of PDZRN3 protein levels, especially when investigating expression changes during developmental processes or experimental manipulations

• Immunofluorescence (IF): For visualization of subcellular localization and co-localization studies with interacting proteins such as Dvl3

• Immunohistochemistry (IHC): For tissue-specific expression analysis, particularly useful in developmental studies and pathological investigations

• Immunocytochemistry (ICC): For cellular localization studies in cultured cells

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of PDZRN3 in various samples

When selecting antibodies for these applications, researchers should consider specificity for particular isoforms and cross-reactivity with orthologs if working with non-human models.

How does PDZRN3 participate in cell cycle regulation and apoptosis?

PDZRN3 plays a significant protective role against apoptosis while promoting cellular proliferation:

• PDZRN3 maintains expression of cyclin A2, a critical cell cycle regulator

• Depletion of PDZRN3 in C2C12 myoblasts reduces the proportion of Ki-67-positive cells, indicating decreased proliferation

• PDZRN3 knockdown decreases Akt phosphorylation, suggesting involvement in survival signaling pathways

• When PDZRN3 is depleted, cells become more susceptible to various apoptotic stimuli, including serum deprivation

• The increased apoptotic response in PDZRN3-depleted cells is evidenced by greater amounts of cleaved caspase-3

• PDZRN3 supports DNA damage repair mechanisms through maintenance of Mre11 expression

• Overexpression of cyclin A2 can rescue the proliferation defects and apoptotic susceptibility in PDZRN3-depleted cells

These findings position PDZRN3 as a potential regulatory target in conditions involving aberrant cell cycle control or apoptotic responses.

What methodological considerations are important when using PDZRN3 antibodies for different experimental applications?

When working with PDZRN3 antibodies, researchers should consider several methodological factors:

For Western Blot:

• Protein extraction method: PDZRN3 is primarily cytoplasmic, so standard cytoplasmic extraction protocols are typically effective

• Expected band size: ~120 kDa for the canonical form, with isoforms potentially appearing at different molecular weights

• Blocking conditions: Optimized blocking (typically 5% BSA or non-fat milk) is crucial to minimize background

• Validation controls: Include positive controls from tissues with known high expression (heart or skeletal muscle) and negative controls using PDZRN3-depleted samples

For Immunofluorescence/Immunohistochemistry:

• Fixation method: Paraformaldehyde (4%) is generally suitable; avoid methanol fixation which can disrupt epitope recognition

• Antigen retrieval: May be necessary for formalin-fixed tissues to expose the epitope

• Co-staining considerations: When performing co-localization studies with Dvl3 or other interacting proteins, ensure antibody compatibility (species, detection systems)

• Signal amplification: Consider tyramide signal amplification for low abundance detection

For all applications:

• Antibody validation: Verify specificity using knockdown/knockout controls or competing peptides

• Cross-reactivity: If working with non-human models, confirm cross-reactivity with the species' PDZRN3 ortholog

• Conjugated versus non-conjugated antibodies: Conjugated antibodies (FITC, biotin, HRP) may offer advantages for specific applications but may have different sensitivity profiles

How does PDZRN3 modulate Wnt signaling pathways and what are the experimental approaches to study this interaction?

PDZRN3 regulates Wnt signaling through a complex interaction with Dishevelled (Dvl3), influencing the balance between canonical and non-canonical pathways:

Mechanism:

• PDZRN3 interacts with Dvl3 through its C-terminal PDZ-binding domain (TTV motif) and the PDZ domain of Dvl3

• PDZRN3 ubiquitinates Dvl3 specifically on its DIX domain, which is critical for canonical Wnt signaling

• This ubiquitination appears to attenuate Wnt/β-catenin canonical signaling while promoting the Wnt/PCP non-canonical pathway

Experimental approaches to study this interaction:

• Co-immunoprecipitation assays: To detect physical interactions between PDZRN3 and Wnt pathway components like Dvl3

• Domain mapping experiments: Using truncated constructs (e.g., PDZRN3ΔPDZB, Dvl3ΔPDZ, Dvl3ΔDIX) to identify interaction domains

• Ubiquitination assays: Co-expression of PDZRN3-V5, Dvl3-myc, and HA-tagged Ubiquitin, followed by immunoprecipitation and detection of ubiquitinated species

• Reporter assays:

  • TOP-Flash/luciferase reporter for β-catenin-dependent transcription

  • AP1-responsive luciferase reporter for c-jun-dependent transcription

• Western blot analysis: Monitoring active β-catenin (ABC) and phosphorylated c-jun (p-c-jun) levels as markers of canonical and PCP pathways, respectively

• Gain and loss of function experiments:

  • siRNA knockdown of PDZRN3

  • Lentiviral overexpression of PDZRN3

  • Conditional deletion models (e.g., Pdzrn3 iECKO)

These experimental approaches can help dissect the role of PDZRN3 in developmental processes and pathological conditions where Wnt signaling is implicated.

What is the significance of PDZRN3 in vascular development and what methods are used to investigate its role?

PDZRN3 plays a critical role in vascular development and angiogenesis, as evidenced by several key findings:

• High expression of PDZRN3 in endothelial cells during development

• Co-localization with CD31+ vessels in embryonic yolk sac (E12.5) and brain (E14.5)

• Expression in postnatal retinal and aortic endothelial cells

• Conditional deletion of Pdzrn3 results in vascular defects, particularly in extraembryonic tissues

Research methodologies to investigate PDZRN3 in vascular development:

• Conditional knockout models:

  • Crossing homozygous LoxP-flanked Pdzrn3 allele with inducible Cre expression systems (e.g., Ubc-cre ERT2)

  • Timed tamoxifen administration to achieve temporal control of gene deletion

• Vascular phenotyping methods:

  • Whole-mount immunostaining of tissues (e.g., retina, yolk sac) with endothelial markers

  • Confocal microscopy for 3D visualization of vascular networks

  • Quantitative analysis of vessel parameters (density, branching, diameter)

• Cell-based angiogenesis assays:

  • Tube formation assays using PDZRN3-depleted endothelial cells

  • Migration and proliferation assays to assess endothelial cell functions

  • Co-culture systems with supporting cells to model complex vascular interactions

• Molecular pathway analysis:

  • Assessment of Wnt pathway activation in endothelial cells with modified PDZRN3 expression

  • Evaluation of endothelial-specific gene expression changes following PDZRN3 manipulation

  • Analysis of endocytosis of Frizzled/Dvl3 complexes in response to PDZRN3 alterations

These approaches collectively allow for comprehensive investigation of PDZRN3's role in vascular biology and potential therapeutic targeting in vascular disorders.

How does PDZRN3 contribute to DNA damage repair mechanisms and cell survival?

PDZRN3 influences DNA damage repair processes primarily through regulation of Mre11 expression and associated survival pathways:

Mechanism and experimental evidence:

• PDZRN3 depletion in C2C12 myoblasts reduces the abundance of Mre11, a component of the MRN complex (Mre11-Rad50-Nbs1) essential for DNA double-strand break repair

• The reduction in Mre11 following PDZRN3 knockdown correlates with increased susceptibility to apoptosis

• This mechanism appears to be cyclin A2-dependent, as overexpression of cyclin A2 restores Mre11 expression in PDZRN3-depleted cells

• PDZRN3 depletion also reduces Akt phosphorylation, suggesting impaired activation of pro-survival signaling

Experimental approaches to study PDZRN3 in DNA damage repair:

• DNA damage induction and assessment:

  • Treatment with DNA-damaging agents (e.g., etoposide, radiation) followed by quantification of γH2AX foci formation

  • Comet assay to measure DNA strand breaks in PDZRN3-manipulated cells

  • Immunostaining for repair factors recruitment to damage sites

• Repair kinetics analysis:

  • Time-course studies of DNA damage resolution in control versus PDZRN3-depleted cells

  • Live-cell imaging with fluorescently tagged repair proteins

• Molecular pathway investigations:

  • Analysis of MRN complex formation and activity

  • Assessment of ATM/ATR pathway activation following DNA damage

  • Evaluation of homologous recombination and non-homologous end joining efficiency

• Rescue experiments:

  • Cyclin A2 overexpression to determine rescue of repair defects

  • Mre11 overexpression to bypass PDZRN3 dependency

  • Structure-function analysis using PDZRN3 mutants to identify domains required for DNA repair functions

Understanding PDZRN3's role in DNA damage repair could have implications for cancer research and cellular responses to genotoxic therapies.

What techniques are most effective for validating PDZRN3 knockout or knockdown models?

Proper validation of PDZRN3 genetic manipulation models is critical for experimental rigor. Several complementary approaches should be employed:

For siRNA/shRNA knockdown validation:

• mRNA level verification:

  • Quantitative RT-PCR using primers targeting different exons of PDZRN3

  • RNA-seq to confirm specificity and examine potential compensatory changes in related genes

• Protein level confirmation:

  • Western blot analysis using antibodies targeting different epitopes of PDZRN3

  • Immunofluorescence to assess cellular distribution and expression levels

  • Flow cytometry for quantitative assessment in individual cells

• Functional validation:

  • Decreased ubiquitination of known PDZRN3 substrates (e.g., Dvl3)

  • Altered Wnt pathway activation (β-catenin accumulation, TOP-Flash activity)

  • Phenotypic changes consistent with PDZRN3 deficiency (increased apoptosis, decreased proliferation)

For CRISPR/Cas9 or conditional knockout validation:

• Genomic verification:

  • PCR-based genotyping to confirm targeted modifications

  • Sequencing to verify exact nature of mutations

  • Southern blot for complex genomic rearrangements

• Complete protein loss confirmation:

  • Western blot analysis with antibodies to different PDZRN3 regions

  • Mass spectrometry to confirm absence of truncated proteins

  • Immunostaining of tissues/cells from knockout models

• Rescue experiments:

  • Re-expression of PDZRN3 to restore normal phenotypes

  • Structure-function analysis using different PDZRN3 domains

• Controls for off-target effects:

  • Use of multiple guide RNAs or knockdown strategies

  • Whole-genome sequencing to identify potential off-target modifications

  • Complementation with knockdown-resistant constructs

These rigorous validation approaches ensure that observed phenotypes are specifically attributable to PDZRN3 deficiency rather than off-target effects or incomplete knockdown.

What experimental approaches are optimal for studying PDZRN3's E3 ubiquitin ligase activity?

As an E3 ubiquitin ligase, PDZRN3's enzymatic activity is central to its cellular functions. Several specialized techniques can be employed to investigate this activity:

In vitro ubiquitination assays:

• Reconstituted ubiquitination system:

  • Purified components: E1, E2, PDZRN3 (E3), substrate protein (e.g., Dvl3), ubiquitin, and ATP

  • Detection of ubiquitinated products by Western blot

  • Mass spectrometry to identify specific ubiquitination sites

• RING domain mutant controls:

  • Generation of catalytically inactive PDZRN3 by mutation of critical residues in the RING domain

  • Comparison of ubiquitination efficiency between wild-type and mutant proteins

Cellular ubiquitination assays:

• Co-immunoprecipitation approaches:

  • Co-expression of PDZRN3, target protein (e.g., Dvl3-myc), and tagged ubiquitin (HA-Ub)

  • Immunoprecipitation of the target protein followed by detection of ubiquitinated species

  • Use of different ubiquitin mutants (K48R, K63R) to determine ubiquitin chain topology

• Proteasomal inhibition:

  • Treatment with proteasome inhibitors (MG132, bortezomib) to accumulate ubiquitinated proteins

  • Comparison of substrate stability in presence/absence of PDZRN3 and proteasome inhibitors

Substrate identification methods:

• Proteomics approaches:

  • Tandem Ubiquitin Binding Entities (TUBEs) pulldown followed by mass spectrometry

  • Stable isotope labeling by amino acids in cell culture (SILAC) comparing PDZRN3 wild-type and knockout cells

• Domain mapping:

  • Construction of substrate deletion mutants to identify regions required for PDZRN3 recognition

  • In vitro binding assays to confirm direct interaction

Functional outcomes:

• Ubiquitin chain topology determination:

  • Use of linkage-specific antibodies to determine K48 (degradative) versus K63 (signaling) linkages

  • Ubiquitin remnant profiling by mass spectrometry

• Substrate fate analysis:

  • Cycloheximide chase experiments to assess protein stability

  • Subcellular fractionation to track changes in substrate localization following ubiquitination

These approaches collectively provide a comprehensive assessment of PDZRN3's E3 ligase activity, substrate specificity, and the functional consequences of substrate ubiquitination.

What are the emerging areas of PDZRN3 research with therapeutic potential?

PDZRN3 research has revealed several promising areas with potential therapeutic applications:

• Muscle regeneration and muscular disorders:

  • PDZRN3's role in myoblast survival and proliferation suggests potential applications in muscular dystrophies or age-related muscle wasting

  • Targeting PDZRN3 might enhance muscle repair processes after injury

• Vascular development and angiogenesis-related disorders:

  • PDZRN3's involvement in vascular development indicates potential therapeutic targets for:

    • Pathological angiogenesis in cancer

    • Vascular malformations

    • Ischemic diseases requiring therapeutic angiogenesis

• Wnt signaling modulation:

  • PDZRN3's ability to shift the balance between canonical and non-canonical Wnt pathways offers opportunities for:

    • Cancer therapy, where aberrant Wnt signaling is common

    • Developmental disorders associated with Wnt pathway dysregulation

    • Regenerative medicine applications

• DNA damage repair and genomic stability:

  • PDZRN3's connection to Mre11 and DNA repair suggests potential in:

    • Cancer therapies targeting DNA repair mechanisms

    • Protection against genotoxic stress

    • Age-related genomic instability conditions

Future research should focus on developing specific modulators of PDZRN3 activity and evaluating their effects in disease models, potentially opening new therapeutic avenues for various pathological conditions.

What are the current technical limitations in PDZRN3 research and how might they be addressed?

Despite significant progress, several technical challenges remain in PDZRN3 research:

• Antibody specificity issues:

  • Problem: Current antibodies may not distinguish between PDZRN3 isoforms

  • Solution: Development of isoform-specific antibodies using unique epitopes; validation with knockout controls

• Temporal and spatial control of PDZRN3 function:

  • Problem: Global knockout can mask tissue-specific functions

  • Solution: More refined conditional knockout models; optogenetic or chemical genetic approaches for acute modulation

• Substrate identification challenges:

  • Problem: Comprehensive identification of PDZRN3 ubiquitination substrates remains incomplete

  • Solution: Advanced proteomics approaches; proximity labeling techniques; development of substrate trapping mutants

• Structural insights:

  • Problem: Limited structural information on PDZRN3 domains and their interactions

  • Solution: Structural biology approaches (X-ray crystallography, cryo-EM) to resolve domain interactions and substrate recognition

• Translational gaps:

  • Problem: Limited studies in human tissues and disease contexts

  • Solution: Development of human cell-based models; analysis of PDZRN3 expression and function in patient samples