HNRNPD Antibody

What are the key functions of HNRNPD/AUF1 in cellular processes?

HNRNPD/AUF1 is a multifunctional RNA binding protein that plays critical roles in several cellular processes. It primarily binds to AU-rich elements (AREs) found within the 3'-UTR of many proto-oncogenes and cytokine mRNAs . HNRNPD functions include mRNA decay, telomere maintenance, translation initiation, and mCRD-mediated mRNA turnover . Recent research has expanded our understanding of HNRNPD's role beyond traditional RNA processing to include DNA damage repair pathways. Specifically, HNRNPD has been identified as a novel player in DNA double-strand break (DSB) repair through homologous recombination (HR) . Though HNRNPD binds chromatin independently of DNA damage, upon damage it relocates to γH2Ax foci and participates in the DNA end resection process that is crucial for HR .

To study these functions, researchers should consider experimental approaches that combine both RNA-protein and DNA-protein interaction analyses, including:

• RNA immunoprecipitation to study native RNA targets

• Chromatin immunoprecipitation to assess DNA binding

• Immunofluorescence to track subcellular localization before and after DNA damage

• Functional assays measuring mRNA stability and translation efficiency

What are the different isoforms of HNRNPD and how should antibody selection account for isoform specificity?

HNRNPD exists in four isoforms of different molecular weights (p37, p40, p42, and p45), all produced by alternate splicing of a single transcript . When selecting antibodies for HNRNPD research, researchers must consider which isoform(s) they wish to target, as the functions of these isoforms may differ in specific cellular contexts.

Methodological approach for isoform-specific studies:

• Examine the antibody epitope information to determine which isoforms will be recognized

• For isoform-specific detection, choose antibodies raised against unique exon junctions

• Validate antibody specificity using cell lines with known isoform expression patterns

• Consider using knockout/knockdown systems followed by isoform-specific reconstitution, as demonstrated in research where HNRNPD knockout cells were generated via CRISPR-Cas9 targeting exon 2

What experimental techniques are most effective for studying HNRNPD's interactions with nucleic acids?

HNRNPD interacts with both RNA and DNA, requiring diverse experimental approaches to fully characterize these interactions. Based on the research literature, these techniques have proven effective:

For RNA interactions:

• UV cross-linking and immunoprecipitation - This technique has successfully demonstrated direct HNRNPD binding to HCV IRES RNA

• RNA pull-down assays using biotinylated RNA and purified HNRNPD protein - Effective for mapping binding sites, as shown in studies identifying stem-loop II of HCV 5' NTR as an interaction region

• CLIP-seq (Cross-linking immunoprecipitation followed by sequencing) - For genome-wide identification of RNA binding sites

For DNA/chromatin interactions:

• Chromatin immunoprecipitation - To study HNRNPD association with chromatin

• DNA damage-induced foci formation assays - Using immunofluorescence to monitor HNRNPD relocalization to γH2Ax foci upon DNA damage

• Synthetic DNA structure approaches - Using DNA structures mimicking resection intermediates as baits to identify protein interactions

How does HNRNPD contribute to DNA double-strand break repair, and what methodologies best measure this function?

HNRNPD has been identified as an important factor in DNA double-strand break (DSB) repair through homologous recombination. Research indicates that while HNRNPD binds to chromatin independently of DNA damage, it relocates to γH2Ax foci upon damage . Its silencing impairs critical processes in the DNA damage response cascade, including:

• CHK1 S345 phosphorylation

• The DNA end resection process

• Single-strand DNA formation upon camptothecin treatment

• AsiSI-induced DSB resection

• RPA32 S4/8 phosphorylation

Methodological approaches to measure HNRNPD's role in DSB repair:

For researchers investigating this function, it's crucial to combine multiple methodologies to build a comprehensive understanding. When using HNRNPD antibodies for these studies, ensure they are validated for immunofluorescence applications with minimal background staining to accurately detect relocalization events.

What controls and validation steps are essential when using HNRNPD antibodies in RNA:DNA hybrid resolution studies?

HNRNPD has been implicated in RNA:DNA hybrid (R-loop) resolution during DNA damage repair. When using antibodies to study this process, proper controls and validation are essential:

Critical controls:

• Positive control: Include known R-loop forming regions (e.g., highly transcribed genes)

• Negative control: Use RNase H-treated samples to eliminate RNA:DNA hybrids

• HNRNPD knockout/knockdown verification: Confirm antibody specificity by showing loss of signal

• Reconstitution experiments: Test function restoration using HNRNPD-expressing constructs with PAM-resistant mutations for CRISPR knockout models

Validation methodology:

• DNA:RNA hybrid immunoprecipitation (DRIP) using antibodies specific to RNA:DNA hybrids (like S9.6)

• Complementary technique verification: Combine immunofluorescence with in situ proximity ligation assays

• Functional validation: RNase H1 expression and RNA polymerase II inhibition should rescue the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage

• Co-immunoprecipitation controls: Verify interaction with known partners like heterogeneous nuclear ribonucleoprotein SAF-A

When publishing results, researchers should clearly document these validation steps to ensure reproducibility and establish confidence in the antibody's performance for this specific application.

How can researchers effectively use HNRNPD antibodies to study its role in translation regulation of viral and cellular mRNAs?

HNRNPD has been identified as an important internal ribosome entry site (IRES) trans-acting factor (ITAF) that modulates translation of viral RNAs like HCV and potentially cellular mRNAs. When using HNRNPD antibodies to study these functions, researchers should consider:

Experimental design approaches:

• Reporter systems: Use dicistronic or monocistronic reporter constructs containing the IRES of interest (e.g., HCV IRES)

• HNRNPD manipulation: Apply overexpression, knockdown (siRNA/shRNA), or knockout (CRISPR-Cas9) strategies

• Ribosome profiling: Monitor shifts in HCV mRNA distribution across polysome fractions upon HNRNPD depletion

• Direct interaction studies: Perform UV cross-linking and immunoprecipitation to confirm HNRNPD binding to the RNA of interest

Translational effect quantification:

• In reporter systems, normalize luciferase activities to RNA levels to accurately measure translation efficiency

• Use replicon systems containing viral proteins to study effects in more physiological contexts

• For infection studies, employ infectious clones containing reporter genes fused to viral proteins

For accurate results when studying HNRNPD's effect on translation, researchers must:

• Include controls for RNA levels, as HNRNPD can affect both translation and RNA stability

• Account for potential isoform-specific effects (the p45 isoform shows strongest ITAF activity for HCV IRES)

• Verify antibody specificity for detecting the relevant HNRNPD isoforms

• Consider cell-type specific effects, as HNRNPD's function may vary between cell lines

What are the optimal conditions for using HNRNPD antibodies in different experimental applications?

Based on the available research data, HNRNPD antibodies have been successfully employed in multiple experimental techniques. Here are the optimal conditions for various applications:

Methodological notes:

• For chromatin-associated studies, optimize fixation conditions (typically 1% formaldehyde for 10 minutes)

• When detecting multiple isoforms, use gradient gels (10-15%) for optimal separation

• For co-immunoprecipitation of interacting partners, gentler lysis conditions are preferable

• Blocking with 5% BSA instead of milk may improve results for phospho-specific antibody detection following HNRNPD studies

• For immunofluorescence, permeabilization conditions affect nuclear signal intensity

What troubleshooting approaches are recommended when working with HNRNPD antibodies in chromatin studies?

When studying HNRNPD's association with chromatin and its role in DNA damage response, researchers may encounter several technical challenges. Here are recommended troubleshooting approaches:

Challenge: Poor signal in chromatin fractions
Solutions:

• Optimize extraction buffers - Use specialized chromatin extraction protocols with increasing salt concentrations

• Adjust fixation conditions - Over-fixation can mask epitopes; try reducing formaldehyde concentration or fixation time

• Include phosphatase inhibitors - Critical for preserving damage-induced phosphorylation events

• Test multiple HNRNPD antibodies - Different epitopes may be differentially accessible in chromatin contexts

Challenge: Non-specific bands in Western blots
Solutions:

• Increase blocking stringency - Use 5% BSA with 0.1% Tween-20

• Optimize antibody concentration - Titrate to find minimal concentration giving specific signal

• Include knockout/knockdown controls - CRISPR-Cas9 knockout cells as negative controls

• Use isoform-specific detection strategies - Target unique exon junctions

Challenge: Inconsistent co-localization with γH2Ax foci
Solutions:

• Standardize damage induction - Control dosage and timing of DNA damaging agents

• Sequential immunostaining - Apply primary antibodies sequentially rather than simultaneously

• Optimize image acquisition settings - Use identical exposure settings for comparative analyses

• Quantify co-localization using software - Apply algorithms to eliminate confirmation bias

How should researchers adapt protocols when using HNRNPD antibodies to study different isoforms in various experimental systems?

HNRNPD exists as four isoforms (p37, p40, p42, and p45), requiring careful experimental design when studying isoform-specific functions. Researchers should modify their protocols as follows:

For Western blot analysis:

• Use longer SDS-PAGE gels (preferably gradient gels) to achieve better separation of closely migrating isoforms

• Employ longer run times at lower voltage to enhance resolution

• Consider using isoform-specific antibodies when available, or antibodies targeting common regions to detect all isoforms

• Include positive controls with known isoform expression patterns

For knockdown/knockout studies:

• Design siRNAs targeting specific exons to selectively deplete certain isoforms:

  • Target exon 7 to deplete p42/p45 but retain p37/p40

  • Use siRNAs targeting conserved regions to deplete all isoforms simultaneously

• For CRISPR-Cas9 knockout, target early exons (e.g., exon 2) for complete knockout

• Design reconstitution experiments with PAM-resistant mutants to verify specificity

For functional studies:

• Express individual isoforms in knockout backgrounds to assess isoform-specific functions

• Use tagged constructs (e.g., Flag-tagged p45) for clean immunoprecipitation

• Consider tissue-specific expression patterns when selecting appropriate experimental systems

• For viral translation studies, note that p45 shows the strongest ITAF activity for HCV IRES

How can HNRNPD antibodies be utilized to study the protein's role in R-loop resolution during DNA repair?

HNRNPD has been implicated in resolving RNA:DNA hybrids (R-loops) during DNA repair, a critical process for maintaining genomic stability. Researchers can utilize HNRNPD antibodies to investigate this role through the following approaches:

Experimental strategy:

• DNA damage induction: Treat cells with agents like camptothecin that are known to generate R-loops

• HNRNPD visualization: Use validated antibodies for immunofluorescence to track HNRNPD localization to damage sites

• R-loop detection: Apply S9.6 antibody (specific for RNA:DNA hybrids) in parallel studies

• Co-localization analysis: Assess overlap between HNRNPD, R-loops, and γH2Ax foci

Functional validation experiments:

• HNRNPD depletion: Generate knockout cells using CRISPR-Cas9 or knockdown using siRNA

• R-loop quantification: Measure R-loop accumulation via DRIP-qPCR or immunofluorescence

• Resolution pathway manipulation: Express RNase H1 or inhibit RNA polymerase II to rescue defects

• Interaction studies: Assess HNRNPD association with known R-loop processing factors like heterogeneous nuclear ribonucleoprotein SAF-A

Research has demonstrated that HNRNPD depletion results in increased RNA:DNA hybrids upon DNA damage, and that expression of RNase H1 or RNA polymerase II inhibition rescues the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells . These findings suggest that R-loop resolution is one mechanism by which HNRNPD facilitates the DNA damage response.

What are the critical considerations when designing experiments to investigate HNRNPD's role in viral translation and replication?

HNRNPD has been identified as a modulator of viral translation, particularly for Hepatitis C virus (HCV), making it an important research target for understanding viral pathogenesis. When designing experiments to investigate this role, researchers should consider:

Experimental system selection:

• Reporter constructs: Use dicistronic reporters containing viral IRES elements driving reporter genes

• Replicon systems: Employ subgenomic or full-length viral replicons for more physiological context

• Infectious virus systems: When available, use reporter-tagged infectious clones

HNRNPD manipulation strategies:

• Overexpression: Express individual isoforms (p37, p40, p42, p45) to identify those with strongest effect

• Knockdown: Use siRNA targeting all isoforms or isoform-specific regions

• Knockout: Generate stable CRISPR-Cas9 knockout cell lines for complete depletion

Critical controls and measurements:

• RNA level normalization: Measure viral RNA levels alongside protein expression, as HNRNPD affects both translation and RNA stability

• Polysome profiling: Analyze redistribution of viral RNA across polysome fractions to confirm translation effects

• Direct binding verification: Use UV cross-linking and immunoprecipitation to confirm protein-RNA interactions

• Viral protein quantification: Western blotting for viral proteins and reporter assays should be performed in parallel

Key insights from published research:

• HNRNPD (particularly p45) enhances HCV IRES-dependent translation

• The protein interacts directly with stem-loop II of HCV 5' NTR

• Knockdown of HNRNPD results in increased HCV RNA replication but decreased translation

• In infection models, HNRNPD depletion significantly hampers HCV infection

These findings suggest HNRNPD may function as a switch between viral translation and replication phases, similar to mechanisms described for other viruses.

How should researchers integrate multi-omics approaches with HNRNPD antibody-based studies to comprehensively understand its cellular functions?

As research on HNRNPD expands beyond individual pathways to systems-level understanding, integration of antibody-based studies with multi-omics approaches becomes increasingly valuable. Here's a methodological framework for such integration:

Transcriptomics integration:

• RNA-seq after HNRNPD manipulation: Identify differentially expressed genes following knockout/knockdown

• RIP-seq (RNA immunoprecipitation sequencing): Use validated HNRNPD antibodies to identify direct RNA targets

• CLIP-seq: Map precise HNRNPD binding sites on RNAs with single-nucleotide resolution

• Correlation analysis: Compare HNRNPD binding patterns with changes in mRNA stability and translation efficiency

Proteomics integration:

• IP-MS: Use HNRNPD antibodies for immunoprecipitation followed by mass spectrometry to identify protein interactors

• BioID or proximity labeling: Identify proteins in close proximity to HNRNPD in different cellular compartments

• Comparative proteomics: Analyze protein expression changes in HNRNPD-depleted vs. control cells

• PTM analysis: Examine how DNA damage affects HNRNPD post-translational modifications

Genomics integration:

• ChIP-seq: Map HNRNPD chromatin binding sites genome-wide

• DRIP-seq: Correlate R-loop formation with HNRNPD binding

• DNA damage mapping: Integrate γH2AX ChIP-seq with HNRNPD binding data

• Chromatin accessibility: Compare ATAC-seq profiles before and after HNRNPD depletion

Data integration strategies:

• Multi-factor analysis: Correlate HNRNPD binding with RNA stability, translation efficiency, and R-loop formation

• Network modeling: Build protein-protein and protein-RNA interaction networks centered on HNRNPD

• Pathway enrichment: Identify biological processes overrepresented in HNRNPD-regulated genes

• Temporal analyses: Examine how HNRNPD functions change during cellular stress response timecourse

This integrated approach can help resolve seemingly contradictory functions of HNRNPD, such as its roles in both promoting HCV translation while inhibiting viral replication , or its context-dependent effects on mRNA stability versus translation.

What quality control measures should be implemented when validating new lots of HNRNPD antibodies?

Antibody lot-to-lot variation can significantly impact experimental reproducibility. When validating new lots of HNRNPD antibodies, researchers should implement these quality control measures:

Basic validation tests:

• Western blot comparison: Run side-by-side blots with old and new antibody lots on:

  • Positive control lysates (cells known to express HNRNPD)

  • Negative control lysates (HNRNPD knockout cells generated via CRISPR-Cas9)

  • Multiple cell lines to assess cross-reactivity patterns

• Dilution series: Test multiple antibody concentrations to determine optimal working dilution

• Blocking peptide competition: Confirm specificity by pre-incubating antibody with immunizing peptide

Advanced validation for specific applications:

• Immunoprecipitation efficiency: Compare protein recovery between antibody lots

• Immunofluorescence pattern consistency: Assess nuclear localization and damage-induced foci formation

• ChIP-qPCR reproducibility: Test enrichment at known binding sites

• Cross-reactivity assessment: Test antibody against recombinant HNRNPD isoforms and related hnRNP family members

Documentation requirements:

• Generate validation reports including images of control experiments

• Record lot number, dilution, incubation conditions, and buffer compositions

• Document detection of all four HNRNPD isoforms (p37, p40, p42, p45)

• Maintain positive control lysates as reference standards

For researchers studying HNRNPD's role in DNA repair, additional validation steps should include testing the antibody's ability to detect HNRNPD relocalization to γH2Ax foci after DNA damage , as this is a critical functional readout.

How can researchers distinguish between direct and indirect effects when studying HNRNPD in complex cellular processes?

HNRNPD participates in multiple cellular processes, making it challenging to distinguish direct from indirect effects when using antibody-based detection methods. Researchers should apply these methodological approaches:

Causality establishment strategies:

• Temporal resolution: Perform time-course experiments to establish order of events

  • Example: Track HNRNPD relocalization to damage sites relative to RPA32 phosphorylation

• Rescue experiments: Reconstitute HNRNPD-depleted cells with:

  • Wild-type HNRNPD

  • RNA-binding deficient mutants

  • DNA-binding deficient mutants

  • Isoform-specific constructs

• Domain mapping: Express individual HNRNPD domains to identify minimal functional units

• Direct interaction verification: Use purified components in vitro to confirm direct biochemical activities

Controls for off-target effects:

• Use multiple knockdown/knockout strategies targeting different regions of HNRNPD

• Include non-targeting controls in all experiments

• Validate phenotypes with multiple HNRNPD antibodies targeting different epitopes

• Consider compensatory effects from other hnRNP family members

For HCV translation studies, researchers demonstrated direct effects by:

• Showing direct binding of purified HNRNPD to HCV IRES RNA

• Demonstrating that ribosomal profiles shift when HNRNPD is depleted

• Confirming that effects on viral translation occur independently of effects on viral replication

For DNA repair studies, direct effects were established by:

• Demonstrating HNRNPD relocalization to damage foci

• Showing that HNRNPD knockout directly impairs in vitro DNA resection

• Establishing that RNA:DNA hybrid accumulation in HNRNPD-depleted cells can be rescued by RNase H1 expression

What are the most effective experimental designs for resolving contradictory findings about HNRNPD functions in different cellular contexts?

Research on HNRNPD has revealed seemingly contradictory functions, such as promoting mRNA decay in some contexts while enhancing translation in others. To resolve such contradictions, consider these experimental design strategies:

Contextual variation analysis:

• Cell type comparative studies: Test HNRNPD function across multiple cell types using identical assays

• Stress-dependent analysis: Examine HNRNPD activity under normal conditions versus various stress stimuli (DNA damage, viral infection, etc.)

• Isoform-specific investigation: Systematically test each HNRNPD isoform (p37, p40, p42, p45) in identical experimental settings

• Substrate-specific effects: Compare HNRNPD's activity on different target RNAs or DNA structures

Mechanistic resolution approaches:

• Competitive binding studies: Assess whether DNA damage affects HNRNPD's RNA binding capacity

• Interactome analysis under different conditions: Compare HNRNPD binding partners before and after stress

• Post-translational modification mapping: Identify condition-specific modifications that might alter HNRNPD function

• Subcellular fractionation: Track HNRNPD distribution between nucleus, cytoplasm, and chromatin across conditions

Example resolution framework for HCV translation versus replication contradiction:
The research indicates HNRNPD enhances HCV translation but suppresses viral RNA replication . This apparent contradiction was addressed by:

• Separating translation from replication measurements

• Using both dicistronic and monocistronic reporter systems

• Normalizing protein expression to RNA levels

• Analyzing polysome profiles to directly assess translation efficiency

For DNA repair function studies, researchers might resolve contradictions by:

• Distinguishing between HNRNPD's roles in different repair pathways (HR vs. NHEJ)

• Examining temporal dynamics of HNRNPD recruitment to damage sites

• Investigating the interplay between HNRNPD's RNA binding and DNA repair functions

• Testing whether R-loop resolution is a primary or secondary function in the repair process