POLDIP3 Antibody

What is POLDIP3 and what are its primary functions in cells?

The protein contains two primary domains: multiple AlkB homolog 2 PCNA Interacting Motifs (APIM) located between amino acids 53-124, which interact with PCNA and the POLD2/p50 subunit of Polδ, and an RNA Recognition Motif (RRM) between amino acids 280-351, which belongs to the Aly/REF family of RNA binding proteins . Through these domains, POLDIP3 mediates its diverse cellular functions.

What POLDIP3 variants exist and how can researchers distinguish between them?

Research has identified two main POLDIP3 protein variants in human cell lines:

• POLDIP3α: The full-length variant with 421 amino acids

• POLDIP3β: A shorter variant with 392 amino acids

Additionally, POLDIP3 splicing variants have been identified:

• Variant-1: Contains all exons (wild-type)

• Variant-2: Lacks exon 3

Researchers can distinguish between these variants using specific antibodies in Western blot analysis. Studies have shown that antibodies can be developed that predominantly react with either POLDIP3 variant-1 or variant-2 . Quantitative RT-PCR using primers that specifically amplify each splice variant is another effective approach for distinguishing between the variants at the mRNA level .

What criteria should researchers consider when selecting a POLDIP3 antibody for specific applications?

When selecting a POLDIP3 antibody for research applications, researchers should consider:

• Target specificity: Determine whether the antibody recognizes specific variants of POLDIP3. Some antibodies may be variant-specific, while others detect all POLDIP3 forms. For example, antibodies have been developed that can differentiate between POLDIP3 variant-1 and variant-2 .

• Application compatibility: Confirm the antibody has been validated for your intended application. Available antibodies have been validated for Western blotting and ELISA techniques .

• Species reactivity: Ensure the antibody reacts with your species of interest. Many commercial antibodies react with human POLDIP3 .

• Immunogen information: Consider the immunogen used to generate the antibody. For example, some antibodies are generated using synthetic peptides derived from specific regions of human POLDIP3 (e.g., amino acids 348-397) .

• Validation data: Review available validation data such as Western blot images showing the expected molecular weight band (approximately 46kDa for POLDIP3) .

How can researchers validate a POLDIP3 antibody before using it in critical experiments?

Proper validation of POLDIP3 antibodies involves several methodological approaches:

• Positive controls: Include lysates from cells known to express POLDIP3, such as RAW264.7 cells which have been used to validate commercial POLDIP3 antibodies .

• Negative controls: Use POLDIP3 genetic knockout (KO) cells generated using CRISPR/Cas9 technology as a negative control to confirm antibody specificity .

• Western blot verification: Confirm the antibody detects a band at the expected molecular weight (approximately 46kDa for POLDIP3) . Multiple POLDIP3 variants may be detected at slightly different sizes.

• Knockdown validation: Use siRNA-mediated knockdown of POLDIP3 to verify antibody specificity. Research has demonstrated that cells treated with POLDIP3 siRNA show decreased POLDIP3 variant-1 protein levels that can be detected by specific antibodies .

• Immunoprecipitation: Perform immunoprecipitation followed by mass spectrometry to confirm the antibody is pulling down POLDIP3 and not cross-reacting with other proteins.

What are the optimal conditions for using POLDIP3 antibodies in Western blotting applications?

For optimal Western blotting with POLDIP3 antibodies, the following methodological approach is recommended:

• Sample preparation: Extract proteins from cells using standard lysis buffers containing protease inhibitors to prevent degradation of POLDIP3.

• Protein loading: Load 20-50μg of total protein per lane on SDS-PAGE gels.

• Antibody dilution: For commercial polyclonal antibodies to POLDIP3, use a dilution range of 1:500-1:1000 for Western blotting applications .

• Detection systems: Secondary antibodies conjugated to HRP are commonly used, followed by ECL detection.

• Expected results: POLDIP3 typically appears as a band at approximately 46kDa. When detecting specific variants, POLDIP3α (421 amino acids) and POLDIP3β (392 amino acids) will appear as distinct bands .

• Controls: Include positive controls such as RAW264.7 cell lysates and negative controls such as POLDIP3-KO cell lysates when available .

How can POLDIP3 antibodies be used to study POLDIP3's role in RNA processing and DNA damage response?

POLDIP3 antibodies can be leveraged in several experimental approaches to investigate its dual roles in RNA metabolism and DNA damage response:

• Immunoprecipitation studies:

  • Use POLDIP3 antibodies to immunoprecipitate protein complexes and identify interacting partners involved in RNA processing (transcription, splicing, export) and DNA damage response.

  • This approach has revealed that POLDIP3 interacts with RPA and Tipin, proteins involved in DNA replication and DNA damage response .

• Chromatin immunoprecipitation (ChIP):

  • Apply POLDIP3 antibodies in ChIP experiments to identify genomic regions where POLDIP3 associates with chromatin.

  • This is particularly useful for studying its association with telomeres, as research has shown that POLDIP3 is recruited to ALT (Alternative Lengthening of Telomeres) telomeres during replication stress .

• Immunofluorescence microscopy:

  • Use POLDIP3 antibodies to track subcellular localization changes under different conditions.

  • Research has shown that POLDIP3 variant-1 exhibits nuclear immunoreactivity that decreases in cells with reduced TDP-43 expression .

• RNA-immunoprecipitation (RIP):

  • Employ POLDIP3 antibodies in RIP assays to identify RNA targets of POLDIP3.

  • This approach has been used with TDP-43 antibodies to demonstrate that TDP-43 binds to POLDIP3 mRNA .

• Checkpoint activation studies:

  • Use POLDIP3 antibodies to monitor changes in POLDIP3 phosphorylation status following DNA damage.

  • Research has shown that POLDIP3 can be phosphorylated at serine-42 in response to DNA damage, potentially by ATM and ATR kinases .

How do post-translational modifications affect POLDIP3 function and how can these be investigated with antibodies?

POLDIP3 undergoes several post-translational modifications that likely regulate its diverse functions:

• Phosphorylation sites:

  • Serine-42: Potentially phosphorylated by ATM and ATR in response to DNA damage

  • Serine-63: Potential Chk1 phosphorylation site

  • Other sites phosphorylated by S6K1 and/or RSK during translation

• Investigation approaches:

  • Phospho-specific antibodies: Develop and utilize antibodies that specifically recognize phosphorylated forms of POLDIP3 at different sites.

  • Phosphatase treatments: Treat immunoprecipitated POLDIP3 with phosphatases and observe mobility shifts using standard POLDIP3 antibodies.

  • Mass spectrometry: Use POLDIP3 antibodies to immunoprecipitate the protein, followed by mass spectrometry to identify and quantify post-translational modifications.

  • Mutational analysis: Create phospho-mimetic or phospho-dead mutants of POLDIP3 and examine their function compared to wild-type using POLDIP3 antibodies.

• Functional studies:

  • Use POLDIP3 antibodies in combination with checkpoint inhibitors to determine how phosphorylation affects POLDIP3's role in the DNA damage checkpoint.

  • Combine POLDIP3 antibodies with translation inhibitors to examine how phosphorylation affects POLDIP3's role in translation.

How does POLDIP3 contribute to R-loop metabolism and how can researchers study this function?

POLDIP3 has been implicated in R-loop metabolism, with important implications for genome stability:

• Key research findings:

  • POLDIP3 plays critical roles in disassembling genome-wide R-loop formation

  • POLDIP3 interacts with RTEL1, which may be important for R-loop regulation

  • Telomere-specific R-loops (TERRA R-loops) are more enriched at ALT telomeres and accumulation of these R-loops induces replication stress

• Experimental approaches using POLDIP3 antibodies:

  • DNA-RNA immunoprecipitation (DRIP): Combine with POLDIP3 ChIP to correlate POLDIP3 binding sites with R-loop locations.

  • Proximity ligation assay (PLA): Use POLDIP3 antibodies with antibodies against R-loop markers like S9.6 to detect in situ interactions.

  • Immunofluorescence co-localization: Use POLDIP3 antibodies together with S9.6 antibodies (which recognize R-loops) to examine co-localization under different conditions.

  • Co-IP studies: Use POLDIP3 antibodies to immunoprecipitate complexes, then probe for known R-loop processing factors like RTEL1.

• Cell models for investigation:

  • POLDIP3 knockout cells generated by CRISPR/Cas9 allow comparison of R-loop levels in the presence or absence of POLDIP3

  • ALT cells provide a model system where telomere-specific R-loops (TERRA R-loops) are enriched and can be studied in relation to POLDIP3 function

How is POLDIP3 splicing altered in neurodegenerative diseases and what methodologies are useful to study these changes?

Research has revealed important connections between POLDIP3 splicing and neurodegenerative diseases, particularly those involving TDP-43 dysfunction:

• POLDIP3 splicing alterations:

  • TDP-43 depletion leads to exclusion of exon 3 in POLDIP3 (producing variant-2 instead of variant-1)

  • This splicing change has been observed in spinal motor neurons from patients with Amyotrophic Lateral Sclerosis (ALS)

• Methodological approaches:

  • RT-PCR analysis: Use primers spanning exon 3 to detect inclusion/exclusion events

  • Quantitative RT-PCR: Employ variant-specific primers to quantify the relative abundance of each POLDIP3 splicing variant

  • Laser capture microdissection: Isolate specific cell populations (e.g., motor neurons) for RNA extraction and analysis of POLDIP3 splicing patterns

  • Immunohistochemistry: Use variant-specific antibodies to detect changes in POLDIP3 variant expression in tissue sections

• Research findings table:

What is the functional significance of POLDIP3 variants in cellular stress response and how can this be investigated?

The different POLDIP3 variants appear to have distinct functional properties, particularly in cellular stress responses:

• Functional differences between variants:

  • POLDIP3 variant-1 (including exon 3) is more effective at maintaining the size of SH-SY5Y cells than variant-2 (lacking exon 3)

  • POLDIP3-KO cells show increased sensitivity to replication blockers, suggesting a role in replication stress response

• Investigative approaches:

  • Complementation studies: Express different POLDIP3 variants in POLDIP3-KO cells and assess rescue of phenotypes such as sensitivity to replication blockers

  • Cellular stress assays: Subject cells expressing different POLDIP3 variants to various stressors (e.g., replication blockers, transcription inhibitors) and assess survival, DNA damage, and checkpoint activation

  • Protein interaction studies: Use variant-specific antibodies to compare the interactome of different POLDIP3 variants under normal and stress conditions

  • Phosphorylation analysis: Examine how different stressors affect the phosphorylation status of POLDIP3 variants using phospho-specific antibodies

• Unified model considerations:
  When designing experiments to study POLDIP3 function in stress response, researchers should consider POLDIP3's multiple roles as proposed in the unified model :

  • Recruitment to nascent RNA during transcription

  • Interaction with ERH to regulate transcription of DNA replication, repair, and DDR genes

  • Modulation of alternative splicing

  • Facilitation of mRNA export via the TREX complex

  • Promotion of translation through interactions with eIF4G and CBP80

  • Activation of replication stress checkpoint through interactions with RPA and Tipin

  • Interaction with RTEL1 to disassemble accumulated R-loops

  • Binding to modified PCNA and stimulation of Pol δ activity during repair of stalled replication forks

By methodically investigating these aspects, researchers can better understand the functional significance of POLDIP3 variants in cellular stress responses.