AUF1 Antibody

What is AUF1 and what cellular functions does it regulate?

AUF1, also known as heterogeneous nuclear ribonucleoprotein D (hnRNPD), is an RNA-binding protein that interacts with AU-rich elements within mRNAs, primarily at the 3' untranslated region (3'UTR). AUF1 has four isoforms (p37, p40, p42, and p45) with a canonical protein mass of approximately 38.4 kilodaltons . The protein is primarily localized in both the nucleus and cytoplasm and is widely expressed across multiple tissue types .

AUF1 functions vary depending on the cellular context:

• It can stabilize mRNAs encoding proteins like TWIST-1, Jun-D, and c-myc

• It can destabilize mRNAs encoding inflammatory cytokines like TNF-α, GM-CSF, COX-1, and IL-2

• It plays crucial roles in cellular proliferation, invasion, and angiogenesis in various cancer types

• It contributes to myogenesis by regulating MEF2C expression

• It facilitates Akt phosphorylation and membrane localization

• It modulates inflammatory responses, with AUF1 knockout mice showing higher susceptibility to septicemia due to TNF-α overproduction

What experimental techniques are commonly used with AUF1 antibodies?

AUF1 antibodies are used in multiple experimental techniques to investigate its functions:

• Western Blot: The most common application for detecting AUF1 protein expression and validating knockout/knockdown efficiency

• RNA Immunoprecipitation (RIP): Used to identify mRNAs bound by AUF1, such as Akt, HIF-1α, and MEF2C mRNAs

• Chromatin Immunoprecipitation (ChIP): Used to identify AUF1's association with specific genomic regions, including the MEF2C promoter

• Immunofluorescence (IF): Used to visualize subcellular localization of AUF1

• Immunohistochemistry (IHC): Used to examine AUF1 expression in tissue samples

• ELISA: Used for quantitative detection of AUF1 in samples

How do the different AUF1 isoforms differ in function?

AUF1 exists in four isoforms (p37, p40, p42, and p45) that differ in their functions:

Research has demonstrated that p40 and p45 isoforms specifically mediate Akt phosphorylation, while p37 and p42 do not have this specific function . This indicates that different isoforms may have specialized roles in cellular signaling pathways.

How can I use AUF1 antibodies to investigate cancer progression mechanisms?

AUF1 antibodies are valuable tools for investigating cancer progression mechanisms, particularly in thyroid cancer and osteosarcoma:

For thyroid cancer research:

• Use CRISPR/Cas9 system for AUF1 knockdown in thyroid cancer cell lines (like TPC1 and IHH4)

• Validate knockdown efficiency using Western Blot and Real-time PCR with anti-AUF1 antibodies

• Assess proliferation rate changes using cell counting experiments

• Evaluate colony formation abilities post-knockdown

• Use Transwell assays to measure migration capacity changes

For osteosarcoma research:

• Use qRT-PCR to measure AUF1 and HIF-1α mRNA levels in aggressive versus less aggressive cell lines

• Perform RIP using anti-AUF1 antibodies to identify AUF1-bound mRNAs involved in angiogenesis

• Validate HIF-1α protein level changes using Western Blot after AUF1 silencing

• Investigate the correlation between AUF1 expression and pro-metastatic phenotypes

Research has shown that AUF1 promotes proliferation and invasion of thyroid cancer cells, and its knockdown significantly reduces these capabilities . In osteosarcoma, AUF1 has been found to stabilize HIF-1α mRNA, promoting angiogenesis, suggesting that anti-angiogenic therapies targeting AUF1 could provide effective treatment methods .

What is the recommended protocol for RNA Immunoprecipitation (RIP) with AUF1 antibodies?

RNA Immunoprecipitation (RIP) Protocol for AUF1:

• Cell Preparation:

  • Grow cells to appropriate confluence (usually 80-90%)

  • For some applications, cross-linking with formaldehyde may be used to stabilize protein-RNA interactions

• Cell Lysis and Immunoprecipitation:

  • Use a commercial RNA-binding protein immunoprecipitation kit (such as Millipore)

  • Prepare whole cell lysates from experimental and control cells

  • Use anti-AUF1 antibodies to pull down AUF1-interacting complexes

  • Include IgG antibodies as a negative control for non-specific binding

• RNA Extraction and Analysis:

  • Extract RNA from immunoprecipitates

  • Perform quantitative RT-PCR to detect and quantify specific mRNAs in the precipitates

  • Calculate enrichment by comparing to the input sample and the IgG control

• Validation Controls:

  • Include known AUF1 targets as positive controls (e.g., Akt, HIF-1α, or MEF2C mRNAs)

  • Include non-targets as negative controls

  • Validate findings with knockdown or overexpression of AUF1

This technique has successfully identified several important AUF1 target mRNAs, including HIF-1α (in osteosarcoma cells) , Akt, glutamine fructose-6-phosphate amidotransferase 1, and SIN1 (a component of mTORC2) .

How do I design CRISPR/Cas9 experiments for studying AUF1 function?

CRISPR/Cas9 Protocol for AUF1 Knockdown:

• sgRNA Design:

  • Design multiple sgRNAs targeting different regions of the AUF1 gene

  • In published research, three different gRNAs (KD1, KD2, KD3) have been used successfully

  • Include appropriate controls with empty vectors or non-targeting sgRNAs

• Cell Preparation and Infection:

  • Prepare target cells (e.g., TPC1 or IHH4 thyroid cancer cells) in the logarithmic growth phase

  • Digest cells with trypsin and plate in a six-well format the day before infection

  • Add packaged sgRNA lentiviral particles to CAS9-expressing cells

  • Incubate at 37°C, 5% CO2

  • Change medium after 10 hours of infection

• Selection and Validation:

  • Begin puromycin selection 72 hours after infection

  • Validate knockdown efficiency using:

    • Real-time PCR to measure AUF1 mRNA levels

    • Western Blot to assess AUF1 protein levels

  • Select the most efficient sgRNAs for further experiments (e.g., KD1 and KD2 in the referenced study)

• Functional Analysis:

  • Assess changes in proliferation using cell counting experiments

  • Evaluate colony formation abilities

  • Measure migration/invasion capacities using Transwell assays

This approach has successfully demonstrated that AUF1 knockdown reduces proliferation and invasion capabilities in thyroid cancer cell lines, providing valuable insights into its role in cancer progression .

What is the relationship between AUF1 and signaling pathways like Akt/mTOR?

AUF1 plays a critical role in the Akt/mTOR signaling pathway through several mechanisms:

• Akt Membrane Localization: AUF1 facilitates the localization of Akt to membrane-containing subcellular compartments, which is essential for its phosphorylation. In AUF1-depleted cells, Akt fails to localize to the membrane and remains predominantly cytosolic and unphosphorylated .

• Isoform-Specific Effects: Expression of p40 and p45 isoforms of AUF1 specifically mediates Akt phosphorylation, while p37 and p42 do not have this effect .

• Binding to Key mRNAs: AUF1 binds to the mRNAs of several components of the Akt/mTOR pathway:

  • Akt mRNA (approximately 5-fold enrichment in AUF1 immunoprecipitates)

  • SIN1 mRNA (a component of mTORC2)

  • Glutamine fructose-6-phosphate amidotransferase 1 (GFAT1) mRNA

• Bidirectional Regulation: The relationship appears bidirectional:

  • Conditions that enhance mTORC2 signaling (like acute glutamine withdrawal) augment AUF1 phosphorylation

  • mTOR inhibition abolishes AUF1 phosphorylation

Understanding this relationship has significant implications for cancer research, as the Akt/mTOR pathway is frequently dysregulated in various malignancies.

Why might I see multiple bands in my AUF1 Western Blot?

Observing multiple bands in AUF1 Western Blots is common and can be attributed to several factors:

• Multiple Isoforms: AUF1 exists in four isoforms (p37, p40, p42, and p45) with different molecular weights, which will appear as distinct bands . This is expected and can actually provide valuable information about isoform-specific expression.

• Post-translational Modifications: AUF1 undergoes various post-translational modifications, including phosphorylation, which can alter its migration pattern on SDS-PAGE . Conditions that enhance mTORC2 signaling can augment AUF1 phosphorylation, potentially resulting in additional bands .

• Degradation Products: Improper sample handling or storage can lead to protein degradation, resulting in smaller fragments detected by the antibody.

• Non-specific Binding: Some antibodies may cross-react with other proteins, particularly other heterogeneous nuclear ribonucleoproteins (hnRNPs) with similar structures.

Recommendations:

• Use positive controls with known AUF1 expression patterns

• Include samples with AUF1 knockdown to identify specific bands (as done in studies using CRISPR/Cas9)

• Consider using isoform-specific antibodies if studying particular AUF1 variants

• Optimize sample preparation and handling to minimize degradation

• Include phosphatase treatment if interested in distinguishing phosphorylated forms

What controls should I include in AUF1 knockdown experiments?

When designing AUF1 knockdown experiments, include these essential controls:

• Non-targeting Controls:

  • Cells infected with empty vector for CRISPR/Cas9 experiments

  • Non-specific siRNA or morpholino for other knockdown approaches

• Knockdown Validation Controls:

  • Real-time PCR to confirm reduction in AUF1 mRNA levels

  • Western Blot to validate protein-level knockdown

  • Test multiple knockdown constructs (e.g., KD1, KD2, KD3) to control for off-target effects

• Functional Controls:

  • Rescue experiments by re-expressing AUF1 to confirm phenotype specificity

  • If studying a specific downstream target (like MEF2C), include experiments restoring that target to determine if it rescues the AUF1 knockdown phenotype

• Target Validation Controls:

  • When studying AUF1's effect on a specific target (like HIF-1α or Akt), measure both mRNA and protein levels of the target in control and knockdown conditions

  • For RNA binding studies, include control immunoprecipitations with non-immune serum or IgG antibodies

These controls help ensure that observed phenotypes are specifically due to AUF1 depletion rather than off-target effects or experimental artifacts, improving the reliability and reproducibility of your findings.

How do I optimize subcellular fractionation for AUF1 studies?

Subcellular fractionation is crucial for studying AUF1's functions, as its localization affects its activity. Here's how to optimize this technique:

• Fractionation Protocol:

  • Separate cellular components into cytosolic and membrane-containing fractions (HSP - heavy/light membrane and soluble pellet)

  • For AUF1-Akt studies, this separation is critical as Akt phosphorylation occurs at membrane compartments

• Marker Validation:

  • Confirm fractionation quality using compartment-specific markers

  • For cytosolic fraction: Use markers like GAPDH or β-actin

  • For membrane fraction: Use markers like Na+/K+ ATPase

  • For nuclear fraction: Use markers like Lamin B1 or histone proteins

• AUF1 Detection Optimization:

  • Use freshly prepared samples to avoid degradation

  • Adjust lysis buffer conditions to effectively extract AUF1 from different compartments

  • Consider crosslinking if studying protein-RNA interactions that might be transient

  • Validate with immunofluorescence to confirm localization patterns

• Analysis Considerations:

  • Compare localization patterns between wild-type and experimental conditions

  • In AUF1 knockdown studies, analyze how target protein localization changes (e.g., Akt shifts from membrane to cytosolic fractions)

  • Correlate localization with phosphorylation status for signaling proteins

In published research, subcellular fractionation revealed that Akt fails to localize to membrane compartments in AUF1-depleted cells, remaining primarily cytosolic and unphosphorylated . This finding was crucial for understanding AUF1's role in facilitating Akt phosphorylation.

How can I use AUF1 antibodies to study its role in inflammatory diseases?

AUF1 has significant implications in inflammatory diseases due to its regulation of cytokine mRNA stability:

• Experimental Models:

  • Study AUF1 knockout mice, which show higher susceptibility to septicemia due to TNF-α overproduction

  • Use cell-penetrating morpholinos for targeted AUF1 knockdown in specific tissues

  • Analyze colon biopsy samples from inflammatory bowel disease (IBD) patients treated with butyrate, which activates AUF1

• Technical Approaches:

  • Use mRNP immunoprecipitation with AUF1 antibodies to identify inflammatory cytokine mRNAs regulated by AUF1

  • Perform quantitative RT-PCR to measure stability of target mRNAs (TNF-α, IL-2, GM-CSF) in the presence or absence of AUF1

  • Use Western Blot to assess how inflammatory stimuli affect AUF1 phosphorylation status

  • Investigate butyrate-mediated effects on AUF1 activation and subsequent changes in inflammatory markers

• Analytical Considerations:

  • Compare AUF1-bound mRNA profiles between healthy and inflamed tissues

  • Assess how AUF1 phosphorylation status changes during inflammatory responses

  • Correlate AUF1 levels with disease severity markers

Recent research has shown that butyrate ameliorates inflammation in colon biopsy samples from IBD patients, partially through activation of AUF1, which regulates cytokine mRNA stability . This suggests potential therapeutic approaches targeting AUF1 for inflammatory diseases.

What are promising new applications for AUF1 antibodies in research?

Current research suggests several promising future directions for AUF1 antibody applications:

• Therapeutic Target Validation: As anti-angiogenic therapies targeting AUF1 show promise for treating osteosarcoma , antibodies can help validate AUF1 as a therapeutic target in preclinical models.

• Multi-objective Antibody Design: Emerging computational approaches like AbNovo, which leverages constrained preference optimization for multi-objective antibody design , could be applied to develop more specific AUF1 antibodies with improved properties.

• Tissue-Specific AUF1 Functions: Investigating tissue-specific roles of AUF1 isoforms in development and disease, building on findings that AUF1 knockout mice display altered skeletal and muscular systems .

• RNA-Protein Interaction Maps: Using advanced techniques like PAR-CLIP (Photoactivatable-Ribonucleotide-Enhanced Cross-Linking and Immunoprecipitation) with AUF1 antibodies to map RNA-protein interactions at high resolution .

• Inflammatory Disease Biomarkers: Exploring AUF1 as a biomarker for inflammatory diseases, given its role in regulating TNF-α and other cytokines .