DHX36 Antibody

What is DHX36 and why is it important in cellular biology?

DHX36 (also known as G4R1 or RHAU) is a multifunctional ATP-dependent helicase that specifically unwinds G-quadruplex (G4) structures in both DNA and RNA. It plays crucial roles in genomic integrity, gene expression regulation, and antiviral responses . The protein's ability to resolve G4 structures makes it particularly important for maintaining cellular function, as these structures can impede DNA replication, transcription, and translation. DHX36 is predominantly localized in the cytoplasm and actively interacts with polyadenylated RNA, suggesting its primary role in post-transcriptional regulation .

Which applications can DHX36 antibodies be reliably used for?

DHX36 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Reliable detection at 1:500-1:2000 dilution

• Immunoprecipitation (IP): Effective at 0.5-4.0 μg for 1.0-3.0 mg total protein lysate

• Immunohistochemistry (IHC): Functional at 1:50-1:500 dilution

• RNA Immunoprecipitation (RIP): Validated in multiple publications

• Co-immunoprecipitation (CoIP): Confirmed for protein-protein interaction studies

• Immunofluorescence (IF): Validated for subcellular localization studies

What is the recommended protocol for DHX36 antibody validation in knockout/knockdown experiments?

For robust validation, researchers should:

• Generate DHX36 knockdown (KD) or knockout (KO) cell lines using RNA interference (shRNA) or CRISPR-Cas9 technologies

• Confirm knockdown efficiency by quantitative PCR and Western blot analysis

• Run parallel immunoblots with wildtype and KD/KO samples under identical conditions

• Include appropriate loading controls

• Compare band intensity at the expected molecular weight (110-120 kDa)

Research shows that doxycycline-inducible DHX36 knockdown systems can achieve significant protein reduction after 96 hours of treatment, as demonstrated by immunoblot analysis . This approach provides a valuable control for antibody specificity verification.

How should researchers optimize DHX36 antibody conditions for studying G-quadruplex structures?

When investigating G-quadruplex (G4) structures with DHX36 antibodies:

• Sample preparation: Use chemical crosslinking (like formaldehyde) to preserve protein-nucleic acid interactions

• G4 stabilizing conditions: Include potassium ions (K+) in buffers to stabilize G4 structures

• Controls: Include parallel experiments with:

  • G4-forming sequences (positive control)

  • Non-G4 sequences (negative control)

  • DHX36 wildtype and mutant (e.g., DHX36ΔRSM or DHX36AAA) samples

• Validation methods: Combine antibody approaches with G4-specific detection methods like BG4 antibody staining or small molecule G4 ligands

Research indicates that DHX36 exhibits much higher binding and enzymatic activities for G4-DNA compared to unstructured DNA forms, making proper experimental controls critical .

What are the important considerations when using DHX36 antibodies in neurological disease models?

When applying DHX36 antibodies in neurological disease research:

• Sample selection: Select appropriate tissue regions based on disease pathology (e.g., medial prefrontal cortex for fear conditioning studies)

• Timing considerations: DHX36 expression can vary temporally after learning or stress events (significant increase observed at 5h post-training in fear conditioning)

• Cell-type specificity: Consider cell type-specific analysis as DHX36 distribution depends on neuronal activation states

• Disease models: For C9orf72-related ALS/FTD research, assess both RAN translation and G4 structure formation using complementary approaches

• Validation: Confirm findings in patient-derived cells with endogenous repeat expansions alongside model systems

Studies show DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, offering therapeutic implications for nucleotide repeat expansion disorders .

How can DHX36 antibodies be used to investigate stress response mechanisms?

For stress response investigations:

• Stress granule detection: Co-stain with DHX36 antibody and stress granule markers (G3BP1, TIA-1) before and after stress induction

• Integrated stress response: Monitor DHX36 during ISR activation using:

  • Thapsigargin or tunicamycin treatment

  • Phospho-eIF2α co-staining

  • puromycin incorporation assays for global translation assessment

• Subcellular fractionation: Perform cytoplasmic/nuclear fractionation followed by immunoblotting to track stress-induced relocalization

• RIP-seq workflow:

  • Crosslink cells during stress response

  • Immunoprecipitate with DHX36 antibody

  • Sequence associated RNAs

  • Analyze G4-forming potential and stress-responsive mRNAs

Research indicates DHX36 knockout results in increased stress granule formation and protein kinase R (PKR/EIF2AK2) phosphorylation, suggesting its role in resolving rG4-induced cellular stress .

What methodological approaches should be used to study DHX36's interaction with replication protein A (RPA) using antibodies?

To investigate DHX36-RPA interactions:

• Co-immunoprecipitation strategy:

  • Express FLAG-tagged RPA1, RPA2, or RPA3 in cells

  • Prepare lysates under non-denaturing conditions

  • Immunoprecipitate with anti-FLAG antibody

  • Detect DHX36 in immunoprecipitates by western blotting

• Proximity ligation assay:

  • Fix cells during various cell cycle stages

  • Incubate with DHX36 antibody and anti-RPA antibodies

  • Perform proximity ligation following manufacturer protocols

  • Quantify interaction signals and correlate with cell cycle phases

• Chromatin immunoprecipitation (ChIP):

  • Crosslink protein-DNA complexes

  • Perform sequential ChIP with DHX36 and RPA antibodies

  • Identify genomic regions where both proteins co-localize

  • Focus analysis on regions prone to G4 formation

Research shows DHX36 coimmunoprecipitates with RPA, suggesting functional interactions during DNA replication and transcription events when G4-DNA can form .

What are common pitfalls in DHX36 immunohistochemistry experiments and how can they be addressed?

Common challenges and solutions include:

• High background signal:

  • Optimize antibody dilution (start with 1:50-1:500 range)

  • For human prostate cancer tissue, use TE buffer pH 9.0 for antigen retrieval

  • Alternative: try citrate buffer pH 6.0 for different tissue types

  • Include proper blocking with BSA or serum

• Inconsistent staining between tissues:

  • DHX36 expression varies between brain regions and cell types

  • Standardize fixation time and conditions

  • Use positive control tissues with known DHX36 expression

  • Consider epitope exposure differences between tissues

• Validation approaches:

  • Include DHX36 knockdown/knockout control tissues

  • Perform peptide competition assays

  • Compare staining patterns with multiple antibodies targeting different DHX36 epitopes

How should researchers interpret contradictory DHX36 antibody data across different cell lines or experimental conditions?

When facing contradictory results:

• Cell-type specific considerations:

  • DHX36 expression and localization can vary significantly between cell types

  • Verify DHX36 expression levels in each cell line by qPCR

  • Consider isoform expression differences that may affect antibody recognition

• Technical validation steps:

  • Confirm antibody specificity in each cell line using knockdown controls

  • Test multiple antibodies targeting different DHX36 epitopes

  • Validate with orthogonal methods (e.g., mass spectrometry)

• Experimental condition effects:

  • DHX36 function can be stress-responsive and context-dependent

  • Document all experimental variables (confluency, passage number, media conditions)

  • Standardize lysate preparation and protein quantification methods

• Data analysis:

  • Normalize to appropriate housekeeping controls for each cell line

  • Consider statistical approaches for handling variability

  • Report all experimental details to facilitate reproduction

How can DHX36 antibodies be utilized to investigate neurodegenerative disease mechanisms?

For neurodegenerative disease research:

• C9orf72 ALS/FTD models:

  • Use DHX36 antibodies in conjunction with dipeptide repeat protein (DPR) antibodies to assess correlation between DHX36 levels and RAN translation products

  • Compare DHX36 binding to C9orf72 repeat RNA using RNA immunoprecipitation followed by qPCR

  • Assess effects of DHX36 modulation on repeat-associated toxicity

• Patient-derived samples:

  • Compare DHX36 levels in patient vs. control tissues

  • Assess co-localization with pathological aggregates

  • Evaluate DHX36 sequestration by disease-associated repeat expansions

• Therapeutic target validation:

  • Monitor changes in DHX36 localization or activity following potential therapeutic interventions

  • Assess DHX36 helicase activity with G4-RNA substrates derived from disease-relevant sequences

Studies indicate that DHX36 depletion suppresses RAN translation from C9orf72 repeat expansions, suggesting it as a potential therapeutic target for G-rich repeat-associated neurological diseases .

What experimental approaches are recommended for studying DHX36's role in DNA damage repair using antibodies?

To investigate DHX36 in DNA damage responses:

• DNA damage induction and assessment:

  • Induce DNA damage with UV, ionizing radiation, or chemical agents

  • Quantify 53BP1 foci formation (marker of DNA double-strand breaks) in control vs. DHX36-depleted cells

  • Research shows DHX36-depleted cells exhibit approximately 30% cells with ≥5 53BP1 foci compared to ~5% in control cells

• Co-localization analysis:

  • Perform immunofluorescence co-staining with DHX36 antibody and DNA damage markers (γH2AX, 53BP1)

  • Analyze recruitment kinetics of DHX36 to damage sites

  • Quantify co-localization coefficients at different time points after damage

• Cell cycle analysis:

  • Synchronize cells and induce damage at specific cell cycle phases

  • Assess DHX36 recruitment to damage sites in relation to cell cycle stage

  • Monitor cell cycle progression following damage in DHX36-proficient vs. deficient cells

• G4-related damage assessment:

  • Use G4 ligands to induce G4-stabilization and analyze DHX36 recruitment

  • Compare damage responses at G4-forming vs. non-G4 genomic regions

Research indicates DHX36 may suppress DNA damage by promoting the clearance of G4-DNA structures, supporting cell growth and survival .

What is the recommended workflow for identifying DHX36 RNA targets using RIP-seq techniques?

For comprehensive RIP-seq analysis:

• Experimental setup:

  • Crosslink cells with formaldehyde or UV

  • Lyse cells and fragment RNA to optimal size range

  • Immunoprecipitate with DHX36 antibody (0.5-4.0 μg per IP reaction)

  • Include IgG control IP and input samples

• RNA processing and sequencing:

  • Extract RNA from immunoprecipitates

  • Verify enrichment by qRT-PCR of known targets

  • Prepare libraries and perform deep sequencing

  • Analyze data for G-rich motifs and G4-forming potential

• Bioinformatic analysis:

  • Identify enriched transcripts compared to input and IgG controls

  • Analyze for G-quadruplex forming sequences (G3+N1-7)n pattern

  • Perform Gene Ontology analysis on target transcripts

  • Compare with published DHX36 targets

Research has identified >4500 mRNAs as DHX36 targets, showing preferential interaction with G-rich and G4-forming sequences, and DHX36 knockout resulting in increased target mRNA abundance but decreased ribosome occupancy .

How can researchers assess the impact of DHX36 on global translation using antibody-based approaches?

To evaluate DHX36's impact on translation:

• SUnSET (Surface Sensing of Translation) assay:

  • Treat control and DHX36-knockdown cells with puromycin (10 minutes)

  • Detect puromycin incorporation by immunoblotting

  • Quantify signal intensity across molecular weight range

  • Research shows no difference in global translation rates between DHX36 control and knockdown cells

• Polysome profiling:

  • Fractionate cytoplasmic lysates on sucrose gradients

  • Collect fractions and analyze by western blotting for DHX36

  • Extract RNA from each fraction for specific target analysis

  • Compare polysome association of G4-containing vs. control mRNAs

• Ribosome profiling with DHX36 modulation:

  • Generate libraries from ribosome-protected fragments in control vs. DHX36-depleted cells

  • Analyze translational efficiency of G4-containing mRNAs

  • Investigate ribosomal pausing at G4 motifs

• Target-specific translation assays:

  • Use luciferase reporters containing G4 motifs

  • Compare translation efficiency in DHX36 wildtype vs. knockdown conditions

  • Normalize to non-G4 control reporters

Studies demonstrate that while DHX36 knockout increases target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting they become translationally incompetent .

How can DHX36 antibodies be used to study learning-dependent changes in G-quadruplex structures in the brain?

For learning and memory studies:

• Behavioral paradigm setup:

  • Subject animals to learning tasks (e.g., fear conditioning or extinction training)

  • Collect brain tissue at specific timepoints (immediate, 1h, 5h post-training)

  • Research shows significant increase in DHX36 expression 5h post-fear conditioning and after fear extinction learning

• Brain region-specific analysis:

  • Process medial prefrontal cortex (mPFC) or other relevant regions

  • Perform DHX36 immunohistochemistry and western blotting

  • Compare expression levels between trained and control animals

• G4-DNA immunoprecipitation:

  • Perform G4-DNA IP followed by sequencing

  • Analyze distribution patterns (5' UTR, proximal TSS, CDS, 3' UTR)

  • Compare G4-DNA profiles between retention control and extinction-trained animals

• In vivo intervention:

  • Infuse DHX36 shRNA into the mPFC to knockdown expression

  • Assess behavioral outcomes and G4-DNA distribution

  • Compare with scrambled control shRNA

Research demonstrates that DHX36 mRNA expression is transiently induced by both fear conditioning and extinction learning, suggesting its role in learning-dependent genomic regulation .

What methodological considerations are important when investigating DHX36's role in viral RNA sensing using antibodies?

For studying DHX36 in antiviral responses:

• Viral infection models:

  • Select appropriate viral systems (RNA virus preferred)

  • Monitor DHX36 localization before and after infection

  • Assess co-localization with viral RNA using immunofluorescence and in situ hybridization

• Multi-helicase-TICAM1 complex analysis:

  • Immunoprecipitate DHX36 from infected cells

  • Analyze co-precipitation of TICAM1 and other complex components

  • Compare complex formation in response to different viral stimuli

  • DHX36 functions as a component of a multi-helicase-TICAM1 complex that acts as a cytoplasmic sensor of viral double-stranded RNA

• Signaling pathway investigation:

  • Monitor pro-inflammatory cytokine induction in control vs. DHX36-depleted cells

  • Assess phosphorylation of downstream signaling components

  • Correlate with viral replication efficiency

• G4-viral RNA interaction:

  • Analyze viral sequences for G4-forming potential

  • Perform RNA immunoprecipitation to detect DHX36-viral RNA interactions

  • Use G4-stabilizing ligands to assess effects on DHX36-mediated sensing

What statistical approaches are recommended for analyzing DHX36 binding site characteristics from ChIP-seq or RIP-seq data?

For robust computational analysis:

• Binding site identification:

  • Use peak calling algorithms optimized for G-rich regions

  • Apply appropriate background models

  • Consider G-content normalization to avoid bias

• Motif analysis:

  • Search for G-quadruplex consensus sequences (G3+N1-7)n

  • Analyze for other structural elements beyond primary sequence

  • Compare with known G4 databases

• Cross-validation approaches:

  • Correlate DHX36 binding sites with G4 structures identified by:

    • G4-seq

    • BG4 ChIP-seq

    • G4-selective chemical probing

  • Validate a subset of targets using orthogonal techniques

• Functional analysis of targets:

  • Categorize targets by:

    • Binding location (5' UTR, CDS, 3' UTR)

    • Effect on target abundance

    • Translational efficiency

  • Research shows DHX36 binding to the UTRs conferred a considerably stronger effect on mRNA abundance compared to CDS binding sites

How should researchers interpret changes in DHX36 target mRNA abundance versus translational efficiency in knockdown studies?

When analyzing seemingly contradictory effects:

• Integrated analysis approach:

  • Compare RNA-seq data (total RNA abundance) with ribosome profiling data (translational efficiency)

  • Categorize targets based on relationship between abundance and translation

  • Research shows DHX36 knockout leads to increased target mRNA levels but decreased ribosome occupancy

• Mechanistic interpretation framework:

  • DHX36 binding to G4 structures can have dual effects:

    • Resolution of G4s in 5' UTRs may enhance translation initiation

    • Resolution of G4s may also decrease RNA stability

  • Consider subcellular localization of affected transcripts

  • Analyze stress granule association of targets

• G4 structure prediction:

  • Correlate effects with predicted G4 stability

  • Consider G4 location within the transcript

  • Analyze for alternative secondary structures

• Data visualization:

  • Create scatterplots of mRNA abundance vs. translational efficiency

  • Identify outlier transcripts for further mechanistic studies

  • Group targets by shared biological pathways

Research demonstrates that loss of DHX36 results in target mRNAs with G-rich structures becoming translationally inactive despite increased abundance, potentially due to stress granule sequestration .

What experimental approaches can determine DHX36's therapeutic potential in repeat expansion disorders?

For therapeutic development research:

• Target validation in disease models:

  • Create cell and animal models expressing pathogenic repeat expansions

  • Modulate DHX36 levels through knockdown, knockout, or overexpression

  • Measure repeat-associated non-AUG (RAN) translation using reporter assays

  • Research shows DHX36 depletion suppresses RAN translation in a repeat length-dependent manner

• Small molecule screening:

  • Design assays to identify compounds that modulate DHX36-G4 interactions

  • Measure effects on:

    • RAN translation efficiency

    • G4 resolution activity

    • Target RNA stability and translation

• Therapeutic delivery approaches:

  • Test antisense oligonucleotides targeting DHX36

  • Evaluate viral vector-mediated DHX36 modulation

  • Assess cell-type specific effects in relevant disease tissues

• Combinatorial therapeutic strategies:

  • Test DHX36 modulation with G4-stabilizing compounds

  • Combine with integrated stress response modulators

  • Evaluate effects on disease-relevant phenotypes

Studies suggest that modulation of G4-resolving helicases like DHX36 represents a candidate therapeutic strategy for G-rich repeat-associated neurological diseases .

How can researchers investigate the crosstalk between DHX36 and other G-quadruplex binding proteins using antibody-based approaches?

To study protein-protein interactions in G4 biology:

• Sequential ChIP (ChIP-reChIP):

  • Perform first immunoprecipitation with DHX36 antibody

  • Elute complexes and perform second IP with antibodies against other G4 binding proteins

  • Identify genomic regions with co-occupancy

  • Correlate with G4 formation potential

• Proximity-based protein identification:

  • Express DHX36 fused to a proximity labeling enzyme (BioID or APEX)

  • Identify biotinylated proteins in G4-rich environments

  • Validate interactions with co-immunoprecipitation

  • Analyze temporal dynamics of interactions

• Competition assays:

  • Assess binding affinity changes in the presence of other G4 binding proteins

  • Determine if relationships are competitive, cooperative, or sequential

  • Measure effects on G4 resolution efficiency

• Functional validation:

  • Create combined knockdowns of DHX36 and other G4 binding proteins

  • Assess synergistic or antagonistic effects on:

    • Target mRNA abundance

    • Translation efficiency

    • G4-associated genomic stability