FXR1 Antibody

What is FXR1 and why is it significant in research?

FXR1 (Fragile X mental retardation syndrome-related protein 1) is a protein encoded by the FXR1 gene in humans. Also known as FXR1p or fragile X mental retardation autosomal homolog 1, this protein belongs to the Fragile X-related family that includes FMR1 and FXR2. FXR1 is a 69.7 kilodalton protein that has gained significant attention due to its diverse biological functions . Research interest in FXR1 has intensified following discoveries of its crucial role in transcriptional regulation and its requirement for proliferation in certain cancer types, particularly those with TP53/FXR2 co-deletion . FXR1 antibodies are essential tools for investigating this protein's expression patterns, interactions, and functions in both normal physiology and disease states.

What are the primary research applications for FXR1 antibodies?

FXR1 antibodies are utilized across multiple experimental techniques in molecular and cellular biology research. The primary validated applications include:

These techniques have been validated across multiple antibody products, with many showing specific reactivity to human, mouse, and rat FXR1 .

How should researchers approach FXR1 antibody validation?

Proper validation of FXR1 antibodies is crucial for ensuring experimental reliability. A systematic validation approach should include:

• Specificity testing: Confirm antibody specificity using positive and negative controls. For FXR1 research, cell lines with known FXR1 expression (positive control) and FXR1 knockdown cells (negative control) are ideal. The H358 and KATOIII cell lines with FXR1 knockdown have been successfully used as negative controls in published research .

• Cross-reactivity assessment: Test for potential cross-reactivity with other Fragile X family members (FMR1 and FXR2) due to sequence homology. While FXR1 and FXR2 show functional redundancy in some contexts, they are distinct proteins that require specific detection .

• Application-specific validation: Each experimental application requires specific validation metrics:

  • For Western blotting: Confirm a single band at the expected molecular weight (~70 kDa for full-length FXR1)

  • For IHC/IF: Include peptide competition assays to verify staining specificity

  • For ChIP: Validate enrichment at known binding sites compared to IgG control

• Isoform consideration: FXR1 exists in multiple isoforms (including full-length isoform a, C-terminal truncated isoform b, and N-terminal tandem Tudor truncated isoform c), so determine which isoforms your antibody recognizes .

How can FXR1 antibodies be utilized to study its role in transcriptional regulation?

Recent research has revealed FXR1's unexpected role in transcriptional regulation. To investigate this function:

• ChIP-seq experiments: Use ChIP-grade FXR1 antibodies to identify genome-wide binding sites. Studies have shown FXR1 locates at gene promoters together with H3K4me3, a marker of active transcription . When designing ChIP experiments:

  • Use appropriate cross-linking conditions (typically 1% formaldehyde for 10 minutes)

  • Include suitable controls (IgG and input chromatin)

  • Consider sequential ChIP (re-ChIP) to study co-occupancy with STAT1/STAT3

• ChIP-MS approach: Combine chromatin immunoprecipitation with mass spectrometry to identify FXR1 protein complexes. This technique has successfully identified multiple FXR1-interacting proteins involved in transcription, including STAT1, STAT3, CHD4, SNF2H, histone H2B, H3K4me3, and DNA topoisomerase TOP2A .

• Correlation with histone modifications: Analyze the co-occurrence of FXR1 binding with specific histone marks. Research has shown significant overlap between FXR1 binding sites and H3K4me3-marked regions, suggesting association with actively transcribed genes .

What methodological approaches can be used to study FXR1's interactions with the STAT pathway?

FXR1 has been shown to interact with STAT1 and STAT3 transcription factors, suggesting an important role in cytokine and growth factor signaling. To investigate these interactions:

• Co-immunoprecipitation: Use FXR1 antibodies for IP followed by Western blot detection of STAT1/STAT3. Research has confirmed interactions between FXR1 and both phosphorylated and unphosphorylated STAT proteins .

• In vitro protein interaction studies: Purified tagged proteins can be used to determine direct interaction. Published experiments using Flag-tagged-FXR1 and HA-tagged-STAT1/3 have demonstrated direct binding .

• Functional studies: Combine FXR1 knockdown with STAT pathway inhibition using:

  • JAK inhibitors like S-Ruxolitinib, which inhibit STAT phosphorylation

  • Gene expression analysis of FXR1 targets following JAK/STAT inhibition

• ChIP-seq integration: Compare binding profiles of FXR1 and STAT1/STAT3. Significant overlap between FXR1, STAT1, and STAT3 ChIP-seq peak-associated genes has been reported (p<1×e-5) .

How can researchers investigate FXR1's role in cancer cell proliferation?

FXR1 inhibition selectively blocks proliferation in cancer cells with TP53/FXR2 co-deletion. To study this function:

• Cell line selection: Use appropriate cellular models:

  • TP53/FXR2 co-deleted lines (H358, HL-60, KATOIII, KMS-11) as experimental models

  • Copy-number-normal cell lines (AGS, A549, MKN45, HepG2) as controls

• FXR1 knockdown approaches:

  • Use validated shRNAs (e.g., FXR1-sh2, FXR1-sh3) that have demonstrated knockdown efficiency

  • Employ inducible knockdown systems to study temporal effects

  • Include rescue experiments with shRNA-resistant FXR1 (FXR1m) to confirm specificity

• Proliferation assessment methods:

  • MTS assay for quantitative measurement

  • Cell imaging and crystal violet staining for visual confirmation

  • Matrigel assays to study three-dimensional growth

• Gene expression analysis: Examine transcriptional changes following FXR1 knockdown, focusing on genes associated with cell cycle and proliferation.

What are critical factors for optimizing FXR1 Western blot protocols?

Western blot optimization for FXR1 detection requires attention to several factors:

• Extraction method: Use RIPA or NP-40 buffer supplemented with protease inhibitors. For nuclear FXR1, include a nuclear extraction step.

• Sample preparation: Heat samples at 95°C for 5 minutes in reducing conditions to ensure proper denaturation.

• Gel selection: Use 8-10% SDS-PAGE gels for optimal separation of the 69.7 kDa FXR1 protein.

• Transfer considerations: Wet transfer at lower voltage (30V) overnight at 4°C may improve transfer efficiency for this higher molecular weight protein.

• Blocking optimization: Test both BSA and non-fat dry milk as blocking agents to determine which provides optimal signal-to-noise ratio.

• Antibody dilution: Start with the manufacturer's recommended dilution (typically 1:1000 for commercially available antibodies) and optimize as needed.

• Detection system selection: Both chemiluminescence and fluorescence-based systems can be effective for FXR1 detection, though fluorescence may offer better quantitative accuracy.

What are the methodological considerations for FXR1 ChIP experiments?

Chromatin immunoprecipitation experiments involving FXR1 require specific technical considerations:

• Chromatin preparation: Generate single-nucleosome chromatin by crosslinking with 1% formaldehyde followed by sonication to fragments of 150-300 bp .

• Antibody selection: Use ChIP-validated FXR1 antibodies that have been tested for specificity and efficiency in chromatin contexts.

• Controls: Include:

  • IgG control to assess non-specific binding

  • Input chromatin (typically 5-10% of starting material)

  • Positive control targets (known FXR1-binding regions)

  • Negative control regions (areas not bound by FXR1)

• Quantitative analysis: For ChIP-PCR validation, use primers targeting potential FXR1 binding regions, particularly promoter regions of genes affected by FXR1 knockdown .

• Sequential ChIP considerations: For co-occupancy studies with STAT proteins or histone marks, optimize antibody concentrations and washing conditions for both immunoprecipitation steps.

How should researchers approach studies of FXR1's Tudor domain interactions?

The tandem Tudor domain of FXR1 is crucial for its function and interacts with histone H3. To study these interactions:

• Domain-specific experiments: Use antibodies that recognize specific domains of FXR1, or express tagged versions of full-length FXR1 (isoform a), C-terminal truncated (isoform b), or N-terminal tandem Tudor truncated (isoform c) proteins .

• Histone interaction studies: Use methyl lysine analog (MLA) proteins with various methylated lysine residues to assess Tudor-histone interactions in pull-down assays. Research has demonstrated interaction between the FXR1 Tudor domain and H3K4me3 .

• Functional rescue experiments: Compare the ability of different FXR1 isoforms to rescue proliferation defects following FXR1 knockdown. Studies have shown that Tudor domain-containing isoforms (a and b) can rescue growth, while Tudor domain-truncated isoform (c) cannot .

• Structural studies: Consider using purified Tudor domains for in vitro binding assays with various modified histone peptides to determine binding specificity and affinity.

How can FXR1 antibodies be integrated into cancer research workflows?

FXR1 antibodies can be strategically incorporated into cancer research through multi-layered approaches:

• Patient sample analysis:

  • Use IHC to evaluate FXR1 expression in tumor tissues

  • Correlate expression with TP53 and FXR2 status

  • Perform survival analysis based on FXR1 expression levels

• Therapeutic target validation:

  • Screen cancer cell line panels with varying TP53/FXR2 status

  • Compare response to FXR1 knockdown across genetic backgrounds

  • Evaluate synthetic lethality in TP53/FXR2 co-deleted contexts

• Mechanism exploration:

  • Identify FXR1 target genes through ChIP-seq and RNA-seq integration

  • Characterize FXR1-dependent transcriptional networks

  • Investigate relationship with STAT signaling pathways

• Translational applications:

  • Develop FXR1 as a potential biomarker for TP53/FXR2 co-deleted cancers

  • Use antibodies to screen patient samples for stratification

  • Monitor FXR1 expression during experimental therapeutic interventions

What emerging applications of FXR1 antibodies should researchers consider?

Several cutting-edge applications of FXR1 antibodies are emerging in contemporary research:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) incorporating FXR1 antibodies

  • Single-cell Western blotting to examine cell-to-cell variability

  • Imaging mass cytometry for spatial context in tissue specimens

• Proximity labeling approaches:

  • BioID or APEX2 fusions with FXR1 to identify proximal proteins

  • Integration with mass spectrometry for comprehensive interactome analysis

  • Validation of proximity results with co-immunoprecipitation using FXR1 antibodies

• Live-cell imaging:

  • FXR1 antibody fragments (Fabs) for live-cell immunofluorescence

  • Nanobody development for dynamic tracking of FXR1

  • Correlation with functional cellular outcomes

• Therapeutic development:

  • Antibody-drug conjugates targeting surface-exposed FXR1 in cancer cells

  • Intrabodies to modulate FXR1 function in specific cellular compartments

  • Screening platforms to identify small molecules that disrupt critical FXR1 interactions

How does FXR1 compare functionally to other Fragile X family members?

Understanding the similarities and differences between FXR1 and related proteins is crucial for proper experimental design:

This comparison is based on experimental evidence showing that FXR2 can partially rescue FXR1 knockdown-induced anti-proliferation, while FMR1 cannot rescue this phenotype and has no impact on proliferation in both TP53/FXR2 copy-number-normal and co-deleted cancer cells .