FUBP3 Antibody

What is the molecular structure and function of FUBP3?

FUBP3 is a 61.6 kDa protein (observed at ~62 kDa in Western blots) that primarily localizes to the nucleus in normal cells. It functions as a transcriptional regulator through binding to single-stranded DNA, particularly at FUSE sites. FUBP3 can also interact with RNA molecules, as evidenced by its binding to FGF9 3'-UTR UG repeats .

Research has demonstrated that FUBP3 plays multiple roles in cellular processes:

• Transcriptional regulation through DNA binding

• Regulation of viral replication, particularly in Japanese Encephalitis Virus (JEV)

• Mediation of amyloid-β-induced neuronal NLRP3 expression

• Potential involvement in cancer progression, particularly in glioblastoma

Under certain conditions like viral infection, FUBP3 can relocalize from the nucleus to the cytoplasm, indicating its function may be regulated by subcellular localization .

How does FUBP3 expression differ between normal and pathological conditions?

• In glioblastoma, FUBP3 is significantly overexpressed compared to adjacent normal brain tissue (p < 0.001)

• In Japanese Encephalitis Virus infection, FUBP3 relocates from the nucleus to the cytoplasm and associates with viral replication complexes

• In Alzheimer's disease models, FUBP3 expression increases with age in the cortex and is required for amyloid-β-induced NLRP3 expression

A comparative study on FUBP3 expression in glioblastoma versus normal tissue revealed:

This data indicates FUBP3 could serve as a potential biomarker for certain pathological conditions .

What are the recommended protocols for FUBP3 detection by Western blot?

For optimal Western blot detection of FUBP3, researchers should follow these methodological guidelines:

Sample Preparation:

• For nuclear protein extraction, use NE-PER™ Nuclear and Cytoplasmic Extraction Reagents following manufacturer's protocol

• Load 15-20 μg of total protein per lane

• Include positive controls such as HeLa, HepG2, MCF7, or HEK-293 cell lysates

Antibody Selection and Dilution:

• Primary antibody dilutions typically range from 1:500-1:5000 depending on the specific antibody

• For polyclonal antibodies: 1:500-1:1000

• For monoclonal antibodies: 1:1000-1:5000

• Blocking buffer: 5% non-fat dry milk in TBST

Detection Parameters:

• Expected molecular weight: approximately 62 kDa

• Secondary antibody: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1:1000-1:5000

When performing Western blot for FUBP3, it's crucial to note the difference between calculated molecular weight (28 kDa) and observed molecular weight (62 kDa) to ensure correct band identification .

What methodologies are effective for FUBP3 knockdown in experimental models?

Several validated approaches for FUBP3 knockdown have been documented in research:

siRNA-Mediated Knockdown:

• Validated siRNA sequence: 5′-GUGUCGAGUAGCUAGC-3′

• Transfection reagent: RNAiMAX lipofectamine in Opti-MEM reduced serum medium

• Protocol: Incubate siRNA with RNAiMAX for 30 min at room temperature prior to transfection

• Validation: Confirm knockdown efficiency after 2-day incubation by Western blotting

shRNA-Mediated Knockdown:

• Validated plasmid: pLKO-fubp-3 shRNA (pFubp-3i: GTGTCGAGTAGCTAGC)

• Control: luciferase shRNA (pNCi: GTACGCGGAATACTTCGA)

• Source: National RNAi Core Facility, Academia Sinica, Taiwan

Viral Vector Delivery for Neuronal Studies:

• For primary neuron cultures: AAV expressing shRNA against FUBP3

• Dosage: 1 × 10^11 genomic copies per well of a 12-well plate

• Application timing: day in vitro (DIV) 4, with media replacement on DIV5

• This approach ensures neuron-specific knockdown in potentially mixed cultures

These methodologies have been validated in different experimental contexts, demonstrating significant reduction in FUBP3 expression and corresponding functional effects on target systems.

How should researchers design protocols for studying FUBP3's interaction with nucleic acids?

When investigating FUBP3's interactions with nucleic acids, researchers should consider the following methodological approaches:

Electrophoretic Mobility Shift Assay (EMSA):

• Use Lightshift Chemiluminescent EMSA kit or equivalent

• For standard EMSA with FUBP3, label probes at 3' ends with biotin rather than 5' ends

• Include competition assays with 1× and 5× unlabeled probes

• For super-shift assays, incubate nuclear extract, labeled probe, and anti-FUBP3 antibody (1 μg) overnight at 4°C

• Test both double-stranded and single-stranded nucleic acids separately

EMSA-Western Blot Approach:

• After EMSA, immerse gels in SDS-PAGE running buffer for 30 minutes to denature proteins

• Transfer to nitrocellulose membrane

• Probe with anti-FUBP3 antibody

• This approach helps detect FUBP3-nucleic acid interactions that may not be visible in standard EMSA

Pull-Down Assays:

• Biotinylated oligonucleotides can be used with avidin-conjugated beads

• This protects biotin labels from potential trimming activity of FUBP3

• Eluted proteins can be analyzed by Western blot or mass spectrometry

Research has demonstrated that FUBP3, like other FUBP family members, may have 5' end trimming activity when binding to nucleic acids, which can affect detection in standard assays . Understanding these technical considerations is essential for accurately characterizing FUBP3's interactions.

How does FUBP3 contribute to viral replication in Japanese Encephalitis Virus?

FUBP3 has been identified as a critical host protein in the Japanese Encephalitis Virus (JEV) life cycle through specific interaction with the viral 3'UTR:

Mechanistic Role:

• FUBP3 specifically associates with the 3'UTR of JEV, which is essential for viral replication, translation, and encapsidation

• During viral infection, FUBP3 relocates from the nucleus to the cytoplasm

• FUBP3 co-localizes with viral proteins in the JEV replication complex

Experimental Evidence:

• Knockdown of FUBP3 significantly decreases JEV viral titer

• In cells with stable FUBP3 knockdown, viral NS5 protein becomes almost undetectable

• Conversely, ectopic overexpression of FUBP3 significantly increases viral RNA production over time compared to controls

• FUBP3 is shown to assist viral replication after JEV infection

Contrast with Other FUBP Family Members:

• While FUBP3 positively regulates JEV replication, FUBP1 has been reported to negatively regulate the JEV infection cycle

• This indicates distinct and potentially opposing roles of different FUBP family members in viral infections

These findings suggest FUBP3 could be a potential target for antiviral therapeutic strategies against JEV infection, highlighting the importance of host-virus interactions in disease pathogenesis.

What is FUBP3's role in neuroinflammation and Alzheimer's disease pathology?

Recent research has identified FUBP3 as a critical transcription factor regulating NLRP3 inflammasome expression in neurons, with significant implications for Alzheimer's disease:

Regulatory Mechanism:

• FUBP3 binds to the minimal NLRP3 promoter and functions as a transcription factor

• This binding was confirmed through multiple methods including EMSA and pull-down assays

• FUBP3 positively regulates NLRP3 expression, particularly in response to amyloid-β (Aβ)

Functional Impact on Neuroinflammation:

• FUBP3 knockdown in Aβ-producing neurons:

  • Reduces NLRP3 protein levels by approximately 88%

  • Decreases secreted IL-1β by approximately 30% (from 7.177 ± 0.6557 pg/mL to 4.817 ± 1.328 pg/mL)

  • Significantly reduces tau phosphorylation at sites p-tau181 and p-tau202/205

Transcriptomic Effects:

• Transcriptome analysis revealed that FUBP3 knockdown in Aβ-treated neurons affected:

  • Expression of both NLRP3 and NLRP6 inflammasome components

  • Reduction in PPP1R1A (inhibitor of protein phosphatase 1)

  • Genes involved in immune system function and signaling pathways

Expression Pattern in Alzheimer's Models:

• In young (3-month-old) wild-type mice, FUBP3 is weakly expressed in cortical neurons

• In APP transgenic mice (Alzheimer's model), FUBP3 expression increases with age

• FUBP3 is preferentially expressed in neurons versus non-neuronal cells

These findings suggest FUBP3 inhibition could represent a potential therapeutic strategy for Alzheimer's disease by reducing NLRP3-mediated neuroinflammation and tau pathology.

How is FUBP3 involved in glioblastoma pathogenesis and immune cell infiltration?

FUBP3 has been identified as a key gene in glioblastoma (GBM) through comprehensive bioinformatic analysis and experimental validation:

Expression Pattern and Prognostic Value:

Interaction with Tumor Microenvironment:

• FUBP3 expression correlates with immune cell infiltration in GBM

• High FUBP3 expression is associated with significantly stronger infiltration of:

  • CD4+ T cells

  • CD8+ T cells

  • CD68+ macrophages

This relationship between FUBP3 expression and immune infiltration suggests FUBP3 may play a role in modulating the tumor immune microenvironment. The intensity of immune cell infiltration in relation to FUBP3 expression is summarized in the following data:

These findings suggest FUBP3 could serve as a potential biomarker and therapeutic target in GBM, particularly considering its relationship with the tumor immune microenvironment .

What are common technical challenges when using FUBP3 antibodies and their solutions?

Researchers working with FUBP3 antibodies may encounter several technical challenges that require specific troubleshooting approaches:

Challenge 1: Discrepancy in Molecular Weight Detection

• Issue: Expected molecular weight (28 kDa) differs from observed weight (~62 kDa)

• Solution: This is normal for FUBP3. Use positive control lysates (HeLa, HepG2, HEK-293) to confirm proper band detection

Challenge 2: Nuclear vs. Cytoplasmic Localization

• Issue: FUBP3 can relocalize from nucleus to cytoplasm under certain conditions

• Solution:

  • Use dedicated nuclear extraction reagents (e.g., NE-PER™)

  • Consider analyzing both nuclear and cytoplasmic fractions separately

  • Perform immunofluorescence to track localization changes

Challenge 3: Detection Issues in Standard EMSA

• Issue: 5'-biotin-labeled probes may not show FUBP3 binding in standard EMSA

• Solution:

  • Use 3'-biotin-labeled probes instead

  • Employ EMSA-western blot techniques

  • This issue may be due to FUBP3's potential 5' end trimming activity

Challenge 4: Cross-Reactivity with Other FUBP Family Members

• Issue: Antibodies may cross-react with related proteins (FUBP1, FUBP2)

• Solution:

  • Validate using FUBP3 knockdown samples

  • Use recombinant monoclonal antibodies for higher specificity

  • Include appropriate negative controls

Challenge 5: Variable Immunohistochemistry Results

• Issue: Inconsistent staining in tissue samples

• Solution:

  • Test different antigen retrieval methods (citrate buffer pH 6.0 or TE buffer pH 9.0)

  • Optimize antibody dilutions (1:20-1:200 range)

  • Use positive control tissues with known FUBP3 expression

How can researchers validate the specificity of their FUBP3 antibodies?

Proper validation of FUBP3 antibodies is essential for ensuring reliable experimental results. Researchers should employ the following validation strategies:

1. Genetic Knockdown/Knockout Controls:

• Implement siRNA or shRNA knockdown using validated sequences:

  • siRNA: 5′-GUGUCGAGUAGCUAGC-3′

  • shRNA: pLKO-fubp-3 (GTGTCGAGTAGCTAGC)

• Compare antibody detection in control versus knockdown samples

• Expect significant reduction in signal with specific antibodies

2. Overexpression Systems:

• Generate cells overexpressing tagged FUBP3 (e.g., Flag-tagged FUBP3)

• Confirm detection with both tag-specific and FUBP3-specific antibodies

• Western blot should show increased band intensity at expected molecular weight (~62 kDa)

3. Multiple Antibody Comparison:

• Test antibodies from different vendors or those targeting different epitopes

• Consistent results across antibodies increase confidence in specificity

• Commercial FUBP3 antibodies target different regions, including N-terminal (aa 38-67) and other epitopes

4. Application-Specific Validation:

• For immunohistochemistry: Include appropriate positive and negative tissue controls

• For Western blot: Include positive control cell lines (HeLa, HepG2, MCF7, HEK-293)

• For immunoprecipitation: Confirm pulled-down proteins by mass spectrometry or Western blot

5. Super-Shift Assays for DNA/RNA Binding Studies:

• In EMSA experiments, include super-shift assays with anti-FUBP3 antibody

• Specific antibodies will cause further shift or depletion of protein-DNA complex

• Include normal IgG as negative control

Thorough validation ensures experimental observations attributed to FUBP3 are specific and reliable, particularly important when investigating novel functions or interactions.

What controls should be included when studying FUBP3 expression or function?

When designing experiments to study FUBP3, researchers should include the following controls to ensure robust and interpretable results:

For Expression Analysis:

• Positive Controls: Include cell lines with confirmed FUBP3 expression (HeLa, HepG2, HEK-293)

• Negative Controls:

  • FUBP3 knockdown or knockout samples

  • For IHC, include tissues known to lack FUBP3 expression

• Loading Controls: Use appropriate nuclear markers (e.g., Lamin B) when analyzing FUBP3, as it's primarily a nuclear protein

• Isotype Controls: For immunostaining, include appropriate isotype antibody controls

For Functional Studies:

• Rescue Experiments: After FUBP3 knockdown, perform rescue with ectopic FUBP3 expression to confirm specificity of observed effects

• Dose-Response Relationships: When possible, use variable levels of FUBP3 knockdown or overexpression to establish dose-dependent effects

• Time-Course Analysis: Include multiple time points to capture dynamic changes, particularly important for viral infection or stress response studies

For Protein-Nucleic Acid Interaction Studies:

• Competition Assays: Include unlabeled probe at 1× and 5× concentrations

• Specificity Controls: Test both sense and antisense strands as probes

• Super-Shift Controls: Include normal IgG alongside FUBP3-specific antibody

• Binding Site Mutations: Create mutated versions of binding sites to confirm sequence specificity

For Mechanistic Studies:

• Pathway Inhibitors: When studying FUBP3's role in specific pathways (e.g., NLRP3 inflammasome), include appropriate pathway inhibitors as controls (e.g., CY-09 or CORM3 for NLRP3)

• Related Family Members: Consider examining other FUBP family members (FUBP1, FUBP2) to distinguish family-wide versus FUBP3-specific effects

Comprehensive controls enhance data reliability and facilitate accurate interpretation of FUBP3's roles in various biological contexts.

What are emerging areas of FUBP3 research beyond its classical role as a transcription factor?

Recent studies have expanded our understanding of FUBP3 beyond its canonical role as a transcriptional regulator, revealing several emerging research areas:

FUBP3 in Neuroinflammation:

• Regulates NLRP3 and NLRP6 inflammasome expression in neurons

• Mediates amyloid-β-induced neuroinflammatory responses

• Influences tau phosphorylation through potential effects on phosphatase inhibitors (PPP1R1A)

• May represent a novel therapeutic target in neurodegenerative diseases

FUBP3 in Viral-Host Interactions:

• Functions as a host factor supporting JEV replication

• Relocates from nucleus to cytoplasm during viral infection

• Interacts with viral RNA structures (3'UTR)

• May represent a potential target for broad-spectrum antiviral strategies

FUBP3 in Tumor Microenvironment Modulation:

• Associated with immune cell infiltration in glioblastoma

• Correlates with CD4+ T cell, CD8+ T cell, and macrophage presence in tumors

• May influence tumor immune surveillance and response to immunotherapy

• Could serve as a prognostic biomarker in certain cancers

FUBP3 in RNA Regulation:

• Interacts with FGF9 3′-UTR UG repeats

• Positively regulates gene expression post-transcriptionally

• May have broader RNA-binding capabilities beyond transcriptional control

• Potentially involved in RNA stability or translation regulation

These emerging areas highlight FUBP3's multifunctional nature and suggest new experimental directions for researchers investigating disease mechanisms and potential therapeutic approaches.

How can researchers distinguish between the functions of FUBP3 and other FUBP family members?

Distinguishing the specific functions of FUBP3 from other FUBP family members (FUBP1 and FUBP2) requires careful experimental design:

Selective Targeting Approaches:

• Use siRNA/shRNA sequences specific to FUBP3 that do not affect other family members

• Validated FUBP3-specific siRNA: 5′-GUGUCGAGUAGCUAGC-3′

• Confirm knockdown specificity by measuring mRNA/protein levels of all family members

Functional Contrast Analysis:

• FUBP1 negatively regulates JEV infection while FUBP3 positively regulates it

• This opposing function can be leveraged to distinguish their roles

• Examine effects of selective knockdown of each family member on the biological process of interest

Binding Site Characterization:

• Different FUBP family members may have distinct binding preferences

• For DNA/RNA interaction studies, perform competition assays with purified recombinant proteins

• Characterize binding motifs using techniques like SELEX or RNA-seq after crosslinking immunoprecipitation

Subcellular Localization:

• Under certain conditions, FUBP family members may show different localization patterns

• Use family-specific antibodies in immunofluorescence studies

• Track changes in localization during cellular processes (e.g., viral infection, stress responses)

Expression Pattern Analysis:

• Examine tissue and cell-type specific expression of different FUBP family members

• In neuronal systems, FUBP3 shows selective expression patterns that may differ from other family members

• Consider developmental and disease-specific expression changes

Understanding the distinct roles of FUBP3 versus other family members is crucial for accurately interpreting experimental results and identifying specific therapeutic targets.

What methodological approaches are recommended for studying FUBP3 in primary neuronal cultures?

When investigating FUBP3 in primary neuronal cultures, researchers should consider these specialized methodological approaches:

Neuronal Culture Preparation:

• Primary neurons can be isolated from rodent embryonic brain tissue

• Ensure high neuronal purity by appropriate isolation techniques

• For Alzheimer's disease studies, neurons can be derived from APP transgenic mice (e.g., APPswe)

Gene Manipulation in Neurons:

• For FUBP3 knockdown: AAV expressing shRNA against FUBP3

• For FUBP3 overexpression: AAV expressing FUBP3-FLAG

• Dose: 1 × 10^11 genomic copies per well of a 12-well plate

• Timing: Add virus on day in vitro (DIV) 4 with media replacement on DIV5

Neuron-Specificity Verification:

• Confirm cell-type specificity of FUBP3 expression by co-staining with neuronal markers (e.g., NeuN)

• FUBP3 is preferentially expressed in neurons versus non-neuronal cells

• This verification is important as primary cultures may contain small numbers of glial cells

Amyloid-β Treatment Protocols:

• Freshly solubilized Aβ 1–42 can be diluted in DMSO

• Final concentration: typically 1 μM

• Incubation time: overnight for optimal effect

• For NLRP3 inhibition studies, include controls with specific inhibitors (CY-09 at 1 μM or CORM3 at 50 μM for 4 hours)

Specialized Analysis Techniques:

• For IL-1β secretion: Use sensitive ELISA methods (can detect low pg/mL levels)

• For tau phosphorylation: Examine specific sites (p-tau181, p-tau202/205)

• For transcriptomic analysis: Compare gene expression profiles with and without FUBP3 manipulation