HNRNPUL1 Antibody

What are the primary experimental applications for HNRNPUL1 antibodies?

HNRNPUL1 antibodies can be utilized across multiple experimental techniques with varying dilution requirements. The polyclonal HNRNPUL1 rabbit antibody has been validated for Western blot (1:1000-1:10000), immunoprecipitation (1:1000-1:10000), immunohistochemistry (1:20-1:200), immunofluorescence (1:20-1:200), flow cytometry, and ELISA applications . For optimal results, researchers should conduct preliminary titration experiments to determine ideal concentrations for their specific experimental system.

For Western blot applications, HNRNPUL1 presents with an observed molecular weight of 90-115 kDa, which is consistent with its predicted mass and potential post-translational modifications . When conducting immunofluorescence, secondary antibodies such as Rhodamine-labeled goat anti-rabbit IgG have been successfully used for visualization of HNRNPUL1 localization in cellular compartments .

Which cell lines and tissue types have been validated for HNRNPUL1 antibody use?

HNRNPUL1 antibodies have been validated across multiple human cell lines and mouse tissues, making them suitable for various experimental models. The following table summarizes validated systems:

The antibody demonstrates cross-reactivity with human, mouse, and rat HNRNPUL1, although other species have not been thoroughly tested . This broad species reactivity makes the antibody suitable for comparative studies across mammalian models.

How should HNRNPUL1 antibodies be stored and handled for optimal performance?

For maintaining antibody integrity and experimental reproducibility, HNRNPUL1 antibodies should be stored according to specific protocols. The polyclonal rabbit antibody is formulated in PBS with 0.1% sodium azide and 50% glycerol at pH 7.3 . This formulation ensures stability during storage and prevents microbial contamination.

Storage recommendations include:

• Maintain at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles that can degrade antibody performance

• Do not aliquot, as this can introduce contamination and stability issues

• Allow the antibody to equilibrate to room temperature before opening the vial

When handling the antibody, appropriate safety precautions should be followed due to the presence of sodium azide, which is toxic and can form explosive compounds in metal plumbing.

How can HNRNPUL1 antibodies be optimized for chromatin immunoprecipitation (ChIP) applications?

While standard applications like Western blot and immunofluorescence are well-established for HNRNPUL1 antibodies, chromatin immunoprecipitation requires additional optimization due to the protein's dynamic association with both RNA and DNA. Research has identified HNRNPUL1 as highly enriched on small nuclear RNA (snRNA) genes, making it a valuable target for ChIP studies investigating transcriptional regulation .

For successful ChIP optimization:

• Perform crosslinking optimization with different formaldehyde concentrations (0.5-1%) and incubation times (5-15 minutes)

• Include RNase treatment controls to distinguish between direct DNA binding and indirect association through RNA

• Use sonication parameters that generate 200-500bp fragments for optimal immunoprecipitation

• Pre-clear chromatin with protein A/G beads to reduce non-specific binding

• Include appropriate negative controls (IgG and non-target regions) and positive controls (known HNRNPUL1 binding sites like snRNA genes)

ChIP-seq analysis has revealed that HNRNPUL1 shows distinctive binding patterns at gene promoters and at sites downstream of transcription termination sites, particularly for histone genes and snRNA genes . This supports the protein's multifunctional role in RNA processing and transcription regulation.

What experimental approaches can effectively measure HNRNPUL1's role in DNA damage response?

HNRNPUL1 plays a critical role in DNA double-strand break (DSB) repair through its interaction with NBS1 as part of the MRE11-RAD50-NBS1 (MRN) complex . To effectively study this function, researchers can employ several complementary experimental approaches:

• Laser microirradiation coupled with live-cell imaging:

  • Transfect cells with fluorescently-tagged HNRNPUL1 constructs

  • Apply laser microirradiation to induce localized DNA damage

  • Track recruitment kinetics of HNRNPUL1 to damage sites in real-time

  • Compare wild-type and mutant HNRNPUL1 recruitment dynamics

• Proximity ligation assays (PLA):

  • Detect and quantify the interaction between HNRNPUL1 and DNA repair proteins (NBS1, MRE11)

  • Compare interaction frequencies before and after DNA damage induction

  • Assess the impact of HNRNPUL1 mutations on protein-protein interactions

• CRISPR-Cas9 knockout models:

  • Generate HNRNPUL1 knockout cell lines as described in HEK293T cells

  • Assess DSB repair efficiency using comet assays or γH2AX immunostaining

  • Complement with rescue experiments using wild-type or mutant HNRNPUL1

• IP-MS following DNA damage induction:

  • Immunoprecipitate HNRNPUL1 from cells before and after DNA damage

  • Identify damage-specific interaction partners by mass spectrometry

  • Validate key interactions with co-immunoprecipitation and Western blotting

These approaches provide complementary data on HNRNPUL1's recruitment, interactions, and functional impact in the DNA damage response pathway.

How can researchers effectively analyze HNRNPUL1's RNA-binding properties?

HNRNPUL1 is a multifunctional RNA-binding protein with several domains involved in RNA interaction, including an RGG domain and a central domain comprising tightly juxtaposed SPRY and dead polynucleotide kinase (dPNK) folds . To comprehensively analyze its RNA-binding properties, researchers should consider these methodological approaches:

• RNA Immunoprecipitation (RIP) followed by sequencing:

  • Immunoprecipitate HNRNPUL1-bound RNAs using validated antibodies

  • Sequence recovered RNAs to identify binding targets

  • This approach has revealed HNRNPUL1's association with U4 snRNA and other targets

• Cross-linking and Immunoprecipitation (CLIP) methods:

  • Enhanced CLIP (eCLIP) has demonstrated HNRNPUL1 enrichment at the 3' end of U2 snRNA

  • Compare binding patterns with other hnRNP proteins as controls

  • Analyze binding motifs and structural preferences

• In vitro binding assays with recombinant protein domains:

  • Express and purify individual domains (RGG, SPRY, dPNK)

  • Perform electrophoretic mobility shift assays with candidate RNA targets

  • Determine binding affinities and specificities for different RNA structures

• Mutational analysis of binding sites:

  • Create point mutations in key RNA binding domains

  • Assess the impact on binding affinity and specificity

  • Link binding properties to functional outcomes in cellular assays

Research has shown that HNRNPUL1 exhibits approximately 40-fold enrichment within the 3' end of U2 snRNA, specifically in a stem loop required for Integrator cleavage accuracy . This indicates a potential preference for specific RNA secondary structures rather than simple sequence motifs.

How should researchers approach studying HNRNPUL1 mutations associated with ALS?

Recent research has identified heterozygous variants of HNRNPUL1 in amyotrophic lateral sclerosis (ALS) patients, including severe truncating mutations like R541X that suggest loss of HNRNPUL1 function may contribute to disease pathogenesis . When investigating these mutations, researchers should employ a comprehensive approach:

• Patient-derived cell models:

  • Obtain lymphoblastoid cell lines from ALS patients with HNRNPUL1 mutations (e.g., A50T or S249N variants)

  • Analyze protein expression, localization, and function in patient cells

  • Compare with age-matched controls without mutations

• Functional characterization of mutant proteins:

  • Clone wild-type and mutant HNRNPUL1 variants into expression vectors

  • Assess effects on known functions (RNA binding, splicing, DNA repair)

  • Investigate protein-protein interactions affected by mutations

• iPSC-derived motor neuron models:

  • Generate induced pluripotent stem cells from patient samples

  • Differentiate into motor neurons for disease-relevant cellular context

  • Evaluate phenotypes such as cytoplasmic mislocalization, RNA processing defects, or stress granule dynamics

• Animal models:

  • Create transgenic models expressing ALS-associated HNRNPUL1 mutations

  • Assess motor function, neurodegeneration, and molecular pathology

  • Test potential therapeutic approaches

HNRNPUL1 mutations in ALS patients should be studied in the context of other RNA-binding proteins implicated in ALS, as research suggests a common etiology with spinal muscular atrophy (SMA) through disruption of small nuclear ribonucleoprotein (snRNP) biogenesis .

What experimental controls are critical when studying HNRNPUL1 depletion effects?

HNRNPUL1 depletion studies require careful experimental design and controls to avoid misinterpretation of results. Several approaches have been used to deplete HNRNPUL1, including siRNA knockdown, CRISPR-Cas9 knockout, and auxin-inducible degron systems . Critical controls include:

• Expression rescue controls:

  • After HNRNPUL1 depletion, reintroduce wild-type protein to confirm phenotype reversal

  • Include domain-specific mutants to identify essential functional regions

  • Research has shown that the RGG domain is essential for HNRNPUL1 activity

• Off-target effect controls:

  • Use multiple depletion methods (siRNA with different sequences, CRISPR with different guide RNAs)

  • In degron systems, include no-doxycycline/auxin controls to account for basal degradation

  • Monitor expression of closely related proteins (e.g., HNRNPUL2) that may have compensatory effects

• Temporal controls:

  • Distinguish between acute and sustained depletion effects

  • For inducible systems, establish appropriate time points to capture primary versus secondary effects

  • Sustained HNRNPUL1 loss leads to reduced snRNA levels and Cajal body loss

• Cell viability and proliferation monitoring:

  • Assess cell survival and proliferation after HNRNPUL1 depletion

  • Control for potential growth disadvantages in long-term experiments

  • HNRNPUL1 elimination with doxycycline/auxin treatment inhibits cell proliferation

When studying HNRNPUL1 depletion effects on splicing, researchers should combine RNA-seq approaches with mechanistic studies focusing on U4-U6 di-snRNP and tri-snRNP formation, as HNRNPUL1 has been shown to play a critical role in these processes .

How can researchers investigate HNRNPUL1's role in Integrator-mediated RNA processing?

HNRNPUL1 plays a critical role in ensuring efficient Integrator-mediated cleavage of nascent RNA downstream of snRNA genes . To investigate this function, researchers should implement these methodological approaches:

• Comparative functional studies with Integrator components:

  • Compare the effects of HNRNPUL1 depletion with depletion of core Integrator subunits (e.g., INTS11)

  • Research has shown that both depletions result in comparable 2-5-fold increases in unprocessed U1, U2, and U4 snRNA transcripts

  • Use mNET-seq (mammalian native elongating transcript sequencing) to analyze nascent RNA processing

• RNA-protein interaction mapping:

  • Use CLIP approaches to map HNRNPUL1 binding sites on snRNAs

  • Focus on the terminal stem loops of snRNAs (U2, U7, U11) where HNRNPUL1 has been shown to bind

  • Mutate these binding sites to assess functional consequences

• Biochemical reconstitution assays:

  • Purify recombinant HNRNPUL1 and Integrator components

  • Perform in vitro cleavage assays with model substrates

  • Test whether HNRNPUL1 enhances Integrator cleavage efficiency or specificity

• Structural studies of HNRNPUL1-RNA complexes:

  • Use structural biology approaches (X-ray crystallography, cryo-EM) to determine how HNRNPUL1 binds terminal hairpins in snRNAs

  • Investigate how this binding might facilitate Integrator recruitment or activity

  • Examine the potential role of the RGG domain in destabilizing RNA secondary structures

Research has shown that HNRNPUL1 binding is centered approximately 25 bases upstream from the Integrator cleavage site in U2 snRNA, similar to the typical distance between a polyadenylation site and cleavage site in pre-mRNA . This suggests a potential mechanistic parallel worth investigating.

What approaches can elucidate the structure-function relationship of HNRNPUL1's domains?

HNRNPUL1 contains multiple functional domains including an RGG RNA-binding domain, a central globular domain comprising tightly juxtaposed SPRY and dead polynucleotide kinase (dPNK) folds, and regions mediating protein-protein interactions . To investigate structure-function relationships, researchers should consider:

• Domain-specific mutational analysis:

  • Generate targeted mutations in specific domains (RGG, SPRY, dPNK)

  • Assess effects on RNA binding, protein interactions, and cellular functions

  • Focus on the interface between SPRY and dPNK domains, which contains amino acid pairs with complementary electrostatic charges

• Structural biology approaches:

  • Complement AlphaFold predictions with experimental structure determination

  • Use X-ray crystallography or cryo-EM to resolve domain structures

  • Pay particular attention to the region around Thr455-Gln486 in the ligand binding pocket, which shows higher flexibility

• Biochemical characterization of dPNK activity:

  • Investigate the binding of 5'-monophosphorylated RNAs and ATP to the dPNK domain

  • Study the mutually exclusive nature of these interactions

  • Examine the antagonistic relationship with XRN2 when overexpressed

• Comparative analysis with related proteins:

  • Compare HNRNPUL1 structure and function with HNRNPU and HNRNPUL2

  • Contrast the dPNK domain with active kinase domains in proteins like PNKP

  • The dPNK domain shares structural homology with mammalian PNKP (RMSD over 82 Cα pairs of 1.1 Å)

Understanding these structure-function relationships is crucial for interpreting the impact of disease-associated mutations and for developing potential therapeutic approaches targeting specific HNRNPUL1 functions.

How should researchers approach investigating HNRNPUL1's role in snRNP biogenesis and recycling?

HNRNPUL1 plays a multifaceted role in snRNP biogenesis and recycling, particularly in the reformation of U4:U6 di-snRNPs for further rounds of pre-mRNA splicing . To effectively study these complex processes, researchers should implement these methodological approaches:

• snRNP assembly assays:

  • Use RNA immunoprecipitation (RIP) with snRNP-specific factors (e.g., Prp31) to assess tri-snRNP formation

  • Compare di-snRNP and tri-snRNP formation in the presence and absence of HNRNPUL1

  • Depletion of HNRNPUL1 has been shown to disrupt di- and tri-snRNP formation

• Cajal body analysis:

  • Monitor Cajal body formation using immunofluorescence for markers like coilin

  • Assess the impact of HNRNPUL1 depletion on Cajal body number and morphology

  • Sustained HNRNPUL1 loss leads to loss of Cajal bodies, critical structures for snRNP assembly

• Protein interaction network analysis:

  • Investigate HNRNPUL1's interactions with key snRNP assembly factors like SART3

  • Use proximity ligation assays or co-immunoprecipitation to confirm interactions

  • Determine whether these interactions are direct or RNA-mediated

• Functional splicing assays:

  • Employ chromatin-associated RNA-seq to identify splicing events altered by HNRNPUL1 depletion

  • Loss of HNRNPUL1 disrupts splicing fidelity, with over a thousand significantly altered splicing events

  • Use minigene reporters to validate specific splicing changes and mechanistic details

These approaches should be integrated to develop a comprehensive understanding of how HNRNPUL1 contributes to the complex process of snRNP assembly, recycling, and function in the context of pre-mRNA splicing.

How can researchers address non-specific binding issues with HNRNPUL1 antibodies?

Non-specific binding is a common challenge when working with antibodies against RNA-binding proteins like HNRNPUL1. To minimize these issues and ensure experimental reproducibility, researchers should implement these methodological approaches:

• Optimized blocking protocols:

  • Test different blocking agents (BSA, milk, commercial blockers) at various concentrations

  • For Western blots, 5% non-fat dry milk in TBST has been effectively used with HNRNPUL1 antibodies

  • For immunofluorescence, blocking with 3% BSA for 30 minutes has shown good results

• Validation with genetic models:

  • Include HNRNPUL1 knockout or knockdown samples as negative controls

  • CRISPR-Cas9 generated knockout HEK293T cells provide an excellent specificity control

  • Compare signal intensity and pattern between wild-type and knockout/knockdown samples

• Cross-reactivity assessment:

  • Test antibody specificity against closely related proteins (HNRNPUL2, HNRNPU)

  • Perform immunoprecipitation followed by mass spectrometry to identify all bound proteins

  • Evaluate species cross-reactivity if working with non-human models

• Signal-to-noise optimization:

  • For immunofluorescence, optimize antibody concentration (starting with 1:50 dilution)

  • Include appropriate controls for secondary antibodies alone

  • Use fluorescent DNA dyes like DAPI as counterstains to confirm nuclear localization

When troubleshooting HNRNPUL1 detection in Western blots, researchers should note that the protein typically presents with a molecular weight range of 90-115 kDa, which may vary depending on post-translational modifications and isoform expression .

What are the key considerations when using HNRNPUL1 antibodies in co-immunoprecipitation studies?

Co-immunoprecipitation (co-IP) is essential for studying HNRNPUL1's interactions with proteins and RNA, but requires careful optimization. Based on validated protocols, researchers should consider these methodological details:

• Lysis buffer optimization:

  • For protein-protein interactions, use buffers containing 150-300 mM NaCl, 0.5% NP-40 or Triton X-100

  • For RNA-dependent interactions, include RNase inhibitors

  • Compare results with and without RNase treatment to distinguish direct versus RNA-mediated interactions

• Antibody orientation:

  • Test both forward (immunoprecipitate HNRNPUL1 and probe for partners) and reverse (immunoprecipitate partners and probe for HNRNPUL1) co-IP approaches

  • For HNRNPUL1 immunoprecipitation, 5 μg of antibody per 2000 μg of cell lysate has been successfully used

• Controls for specificity:

  • Include isotype-matched IgG controls

  • Use HNRNPUL1 knockout cells as negative controls

  • Include input samples (typically 5-10% of IP material) for quantitative comparison

• Detection strategies:

  • For Western blot detection after IP, use a dilution of 1:2000 for the HNRNPUL1 antibody

  • Consider using antibodies raised in different species for IP and detection to avoid heavy/light chain interference

  • For complex interaction networks, couple IP with mass spectrometry analysis

Successful co-IP of HNRNPUL1 has been demonstrated with HeLa cell lysates , making this cell line a good starting point for optimization. When investigating DNA damage-related interactions, researchers should compare results between untreated and DNA damage-induced conditions to identify damage-specific interactions.

How can researchers optimize RNA immunoprecipitation protocols for HNRNPUL1?

RNA immunoprecipitation (RIP) is critical for understanding HNRNPUL1's RNA-binding properties in vivo. Based on successful studies of HNRNPUL1's interactions with specific RNAs like U4 snRNA , researchers should optimize their RIP protocols with these considerations:

• Crosslinking strategies:

  • Compare UV crosslinking (254 nm for direct protein-RNA interactions) with formaldehyde crosslinking (captures indirect interactions)

  • Optimize crosslinking times to balance efficiency with potential damage to epitopes

  • Include non-crosslinked controls to assess background and specificity

• Lysis and immunoprecipitation conditions:

  • Use lysis buffers with RNase inhibitors to prevent RNA degradation

  • Include appropriate detergent concentrations (0.1-0.5% NP-40) to maintain protein solubility while preserving interactions

  • Optimize wash stringency to balance between specificity and sensitivity

• RNA recovery and analysis:

  • Implement careful RNA extraction procedures to maximize recovery

  • For known targets like U4 snRNA, use RT-qPCR for quantitative analysis

  • For discovery of novel targets, couple with RNA-seq

  • Include appropriate normalization controls (input RNA, non-target RNAs)

• Validation strategies:

  • Confirm enrichment of known HNRNPUL1 targets like U2 snRNA, U4 snRNA, and U7 snRNA

  • Compare RIP results with published eCLIP data showing HNRNPUL1 enrichment at the 3' end of U2 snRNA

  • Validate novel interactions with complementary approaches like EMSA or in vitro binding assays

When investigating context-specific interactions, researchers should compare RIP results between different cellular conditions, such as before and after DNA damage induction or in different cell cycle phases, as HNRNPUL1 plays roles in multiple cellular processes including DNA repair and histone gene regulation .