HNRNPU Antibody

What is HNRNPU and what cellular functions does it perform?

HNRNPU, also known as HNRPU, SAFA, and U21.1, is an 825 amino acid protein that is extensively phosphorylated. It functions as a component of ribonucleosomes and is localized in cytoplasmic mRNP granules containing untranslated mRNAs . HNRNPU was originally identified as a component of heterogeneous ribonucleoprotein (hnRNP) complexes and is also known as nuclear scaffold attachment factor A (SAF-A) .

HNRNPU possesses dual binding capabilities - it has a DNA binding domain at the N-terminus and an RNA-binding domain (RGG domain) at the C-terminus. This dual binding ability enables HNRNPU to perform multiple cellular functions including:

• Transcriptional regulation

• Nuclear matrix/scaffold attachment

• Alternative splicing

• mRNA stability control (particularly for inflammatory cytokines like IL-6 and IL-1β)

• Post-transcriptional regulation

Though primarily nuclear, HNRNPU is a nucleocytoplasmic shuttling protein that can be detected in cytoplasmic fractions, suggesting distinct roles in different cellular compartments .

Why do HNRNPU antibodies show different applications across research platforms?

HNRNPU antibodies demonstrate versatility across multiple experimental applications, with each application providing unique insights into protein function, localization, or interactions. Based on validated research data, HNRNPU antibodies can be reliably used in the following applications:

Each application requires specific antibody dilutions and sample preparation techniques to achieve optimal results .

What are the recommended dilution ratios for different experimental applications?

Proper antibody dilution is critical for experimental success, as it affects signal-to-noise ratio, specificity, and reproducibility. The recommended dilutions for HNRNPU antibodies vary by application:

It is important to note that these dilutions serve as starting points, and researchers should optimize the dilution for their specific experimental system to obtain optimal results . Sample-dependent variations may require further titration.

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

Proper storage and handling of HNRNPU antibodies are essential for maintaining their reactivity and specificity. Based on manufacturer recommendations:

HNRNPU antibodies are typically supplied in liquid form containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For optimal preservation:

• Store antibodies at -20°C where they remain stable for one year after shipment

• Aliquoting is typically unnecessary for -20°C storage

• Some antibody preparations (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizer

• Avoid repeated freeze-thaw cycles as they can denature antibodies and reduce activity

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

• For long-term storage beyond one year, consider making small aliquots to minimize freeze-thaw cycles

Proper handling during experiments is equally important:

• Keep antibodies on ice when in use

• Return to -20°C storage promptly after use

• Avoid contamination by using clean pipette tips when handling antibody stocks

How can I optimize CLIP experiments to study HNRNPU RNA binding properties?

An optimized approach based on recent research includes:

• Improved BrdU-CLIP method: This modified protocol produces approximately 10-fold greater yield of pre-amplified CLIP library, resulting in lower duplicate rate of CLIP-tag reads and reduced PCR cycle requirements .

• Subcellular fractionation: Since HNRNPU shuttles between nucleus and cytoplasm, separate CLIP experiments on nuclear and cytoplasmic fractions can reveal compartment-specific binding patterns. Research has shown that cytoplasmic CLIP (cyto-CLIP) identifies more differentially expressed genes than whole-cell CLIP for HNRNPU .

• Controls and validation:

  • Include universal control RNAs

  • Use a non-shuttling RBP (like HNRNPC) as a control

  • Validate binding sites with RNA immunoprecipitation (RIP) assays

  • Confirm subcellular localization by immunofluorescence and biochemical fractionation

• Technical considerations:

  • While radioisotope labeling has been traditional in CLIP, newer methods like eCLIP and irCLIP may be performed without it

  • When first performing CLIP with HNRNPU, it's advisable to check RNA-RBP complex positions using radioisotope labeling before transitioning to non-radioisotope methods

  • Optimize RNase concentration, lysate volume, and antibody selection

This optimized approach has successfully identified HNRNPU binding to the 3'-UTR of IL-6 mRNA, providing new insights into its role in cytokine regulation .

What are the most effective strategies to study cytoplasmic versus nuclear functions of HNRNPU?

HNRNPU predominantly localizes to the nucleus but also has important cytoplasmic functions. Distinguishing between these compartment-specific roles requires specialized experimental approaches:

Recent research has demonstrated that cytoplasmic HNRNPU interacts with specific mRNAs like IL-6, suggesting an important role in controlling mRNA stability in the cytoplasm despite its predominant nuclear localization by immunofluorescence .

How can I validate HNRNPU's direct binding to specific target mRNAs?

Validating direct binding of HNRNPU to specific target mRNAs requires a multi-faceted approach that combines various RNA-protein interaction assays:

• RNA Immunoprecipitation (RIP):

  • RIP assays can confirm that specific mRNAs (e.g., IL-6) are co-immunoprecipitated with HNRNPU but not with control IgG

  • Include housekeeping genes (e.g., RPLP0) as specificity controls

  • Quantitative PCR of immunoprecipitated RNA can demonstrate enrichment of target mRNAs

• Optimized CLIP approaches:

  • Cytoplasmic CLIP (cyto-CLIP) has been shown to identify more HNRNPU direct targets than whole-cell CLIP

  • For example, analysis combining RNA sequencing data from HNRNPU-knockdown cells with cyto-CLIP identified 214 differentially expressed genes as direct targets, compared to only 44 with whole-cell CLIP

• Functional validation:

  • siRNA-mediated knockdown of HNRNPU followed by measurement of target mRNA and protein levels

  • Experimental example: HNRNPU knockdown in HeLa cells significantly decreased IL-6 mRNA levels after PMA/ionophore stimulation, and reduced IL-6 protein secretion

• Subcellular localization analysis:

  • For cytoplasmic functions like mRNA stability control, confirm binding occurs in the cytoplasmic fraction

  • Example: HNRNPU interaction with IL-6 mRNA was specifically observed in the cytoplasmic fraction, not in the nuclear fraction

• Binding site identification:

  • Map the precise binding regions (e.g., 3'-UTR) through deletion constructs and reporter assays

  • Identify specific binding motifs through mutational analysis

This comprehensive approach provides strong evidence for direct, functionally relevant interactions between HNRNPU and target mRNAs in specific cellular compartments.

What controls should be included when studying HNRNPU's role in inflammatory responses?

• Knockdown/expression controls:

  • Include non-targeting siRNA controls alongside HNRNPU siRNA

  • Validate knockdown efficiency by Western blot and RT-qPCR

  • In overexpression studies, use empty vector controls

• Stimulation controls:

  • Include time-course analysis of stimulation (e.g., 2, 4, and 24 hours post-stimulation)

  • Appropriate unstimulated controls under identical conditions

  • Consider multiple stimulation methods (e.g., PMA/ionophore versus LPS) as HNRNPU's effects may be stimulus-specific

• Target specificity controls:

  • Measure multiple cytokines beyond your target of interest

  • Include housekeeping genes (e.g., RPLP0) as negative controls

  • For RIP experiments, include immunoprecipitation with normal IgG as a specificity control

• Subcellular localization controls:

  • Validate subcellular fractionation using established markers:

    • Cytoplasmic marker: GAPDH

    • Nuclear marker: Lamin B1

  • Perform immunocytochemistry to confirm HNRNPU localization before and after stimulation

• Experimental validation controls:

  • Measure both mRNA levels (by RT-qPCR) and protein levels (by ELISA or Western blot)

  • Include positive controls known to be affected by HNRNPU (e.g., IL-6)

  • For cytoplasmic roles, confirm minimal nuclear contamination in cytoplasmic fractions

Research has demonstrated that HNRNPU regulates IL-6 expression in HeLa cells stimulated with PMA and calcium ionophore, with knockdown significantly decreasing both mRNA and secreted protein levels, highlighting its importance in inflammatory responses .

What are the advantages and limitations of different antibody types for HNRNPU detection?

Different types of antibodies have distinct characteristics that affect their performance in various applications. For HNRNPU detection, researchers should consider:

Experimental considerations when choosing antibodies:

• Application-specific selection:

  • For immunoprecipitation in CLIP or RIP: Monoclonal antibodies like sc-32315 have been successfully used

  • For Western blot: Both types work well, with polyclonals offering potentially higher sensitivity

• Verification approaches:

  • Knockdown/knockout validation to confirm specificity

  • Multiple antibodies targeting different epitopes for confirmation

  • Cross-validation with tagged proteins

• Target-specific considerations:

  • HNRNPU's observed molecular weight (120 kDa) differs from calculated weight (91 kDa), requiring antibodies validated to detect the correct band

  • Consideration of potential post-translational modifications

  • Ability to detect both nuclear and cytoplasmic forms

The choice between polyclonal and monoclonal antibodies should be guided by the specific experimental requirements, with consideration of sensitivity, specificity, and application needs.

How should I interpret discrepancies between immunofluorescence and fractionation results for HNRNPU localization?

A common challenge in HNRNPU research is reconciling the predominantly nuclear localization observed by immunofluorescence with the detection of HNRNPU in cytoplasmic fractions. This apparent discrepancy requires careful interpretation:

This discrepancy highlights the importance of using complementary techniques when studying nucleocytoplasmic shuttling proteins like HNRNPU, as different methods may reveal distinct aspects of their biology.

What strategies can optimize HNRNPU antibody performance in challenging experimental contexts?

Optimizing HNRNPU antibody performance in challenging experimental settings requires careful consideration of multiple factors:

• Western Blot optimization:

  • Sample preparation: Complete protein denaturation is crucial as HNRNPU is a large protein (120 kDa observed)

  • Transfer conditions: Extend transfer time or use specialized buffers for high molecular weight proteins

  • Blocking optimization: Test different blocking agents (BSA vs. milk) to reduce background

  • Primary antibody concentration: Titrate within recommended ranges (1:1000-1:10000) for optimal signal-to-noise ratio

  • Secondary antibody selection: Choose high-sensitivity detection systems for challenging samples

• Immunoprecipitation enhancements:

  • Pre-clear lysates to reduce non-specific binding

  • Optimize antibody amount (0.5-4.0 μg per 1.0-3.0 mg protein lysate)

  • Consider protein A vs. protein G beads based on antibody isotype

  • Adjust wash stringency to balance between specificity and yield

  • For RNA-protein interactions, optimize crosslinking conditions

• Immunofluorescence/Immunohistochemistry refinements:

  • Fixation method: Compare paraformaldehyde vs. methanol fixation

  • Antigen retrieval: For IHC, test TE buffer pH 9.0 vs. citrate buffer pH 6.0

  • Permeabilization optimization: Adjust detergent type and concentration

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

  • Mounting media selection: Use anti-fade reagents to preserve signal during imaging

• Flow cytometry considerations:

  • Cell fixation and permeabilization protocol optimization

  • Titrate antibody concentration (starting with 0.20 μg per 10^6 cells)

  • Include appropriate isotype controls

  • Optimize compensation when using multiple fluorophores

  • Consider using cell cycle phase markers when analyzing nuclear proteins

These optimization strategies should be systematically tested and documented to establish reliable protocols for specific experimental contexts, ensuring reproducible results across different applications of HNRNPU antibodies.

How can HNRNPU antibodies be used to investigate its role in disease mechanisms?

HNRNPU has been implicated in various disease processes, and antibodies against this protein serve as valuable tools for mechanistic investigations:

• Inflammatory disorders:

  • HNRNPU regulates expression of inflammatory cytokines including IL-6 and IL-1β

  • Antibody-based techniques can track HNRNPU's interactions with cytokine mRNAs during inflammation

  • CLIP and RIP assays using HNRNPU antibodies can identify direct binding to inflammatory mRNA targets

  • Changes in HNRNPU expression or localization can be monitored during inflammatory responses

• Cancer research:

  • Recent research indicates that proteins encoded by circHNRNPU promote multiple myeloma progression

  • HNRNPU antibodies can be used to:

    • Assess expression levels across cancer types

    • Investigate alterations in subcellular localization

    • Study interaction with cancer-associated RNAs

    • Examine alternative splicing regulation in tumors

• Neurological disorders:

  • RNA-binding proteins like HNRNPU are often associated with neurological diseases

  • Antibodies enable the study of:

    • Protein aggregation patterns

    • Altered nucleocytoplasmic shuttling

    • Dysregulated RNA processing

    • Post-translational modifications in disease states

• Methodological approaches:

  • Immunohistochemistry of patient samples to assess expression patterns

  • Co-immunoprecipitation to identify altered protein-protein interactions

  • ChIP assays to study altered chromatin interactions

  • CLIP-seq to map disease-specific RNA binding profiles

  • Western blot analysis of patient-derived samples to detect altered expression or post-translational modifications

HNRNPU antibodies are thus essential tools for uncovering the diverse roles of this multifunctional protein in pathological processes, potentially leading to new therapeutic targets and biomarkers.

What emerging techniques might enhance our understanding of HNRNPU biology?

Recent technological advances offer new opportunities to study HNRNPU's complex biology:

• Advanced RNA-protein interaction methodologies:

  • Optimized CLIP methods with greater sensitivity for nucleocytoplasmic shuttling proteins

  • Proximity-dependent RNA labeling techniques for spatial transcriptomics

  • Single-molecule imaging to visualize dynamic RNA-protein interactions

  • CRISPR-based RNA targeting to manipulate specific HNRNPU-RNA interactions

• High-resolution localization techniques:

  • Super-resolution microscopy to better visualize nuclear vs. cytoplasmic distribution

  • Live-cell imaging with tagged HNRNPU to track shuttling dynamics

  • Correlative light and electron microscopy for ultrastructural context

  • Lattice light-sheet microscopy for 4D tracking of HNRNPU movement

• Functional genomics approaches:

  • CRISPR screening to systematically identify HNRNPU functional partners

  • RNA-seq combined with HNRNPU perturbation to map global regulatory networks

  • Ribosome profiling to assess translational impacts of HNRNPU regulation

  • Single-cell multiomics to understand cell-type specific functions

• Structural biology integration:

  • Cryo-EM structures of HNRNPU-RNA complexes

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Integrative structural modeling combining multiple data types

  • AlphaFold-based prediction of protein-RNA interaction interfaces

These emerging approaches, when combined with traditional antibody-based methods, promise to provide unprecedented insights into HNRNPU's diverse cellular functions and disease associations. The integration of multiple techniques will be particularly powerful for understanding the compartment-specific roles of this nucleocytoplasmic shuttling protein.