DUX4 Antibody, FITC conjugated

What is DUX4 and why is it a significant research target?

DUX4 (Double Homeobox 4) is a transcription factor that plays a critical role in early embryonic development by activating the first wave of zygotic gene expression. Aberrant expression of DUX4 has significant pathological implications, most notably in facioscapulohumeral muscular dystrophy (FSHD) where its mis-expression in skeletal muscle contributes to disease pathogenesis. Additionally, DUX4 has been implicated in cancer immune evasion mechanisms through its ability to suppress interferon-gamma (IFNγ) signaling pathways. The protein interacts with STAT1 and broadly suppresses expression of IFNγ-stimulated genes by decreasing bound STAT1 and RNA polymerase II recruitment to target gene promoters . These diverse biological roles make DUX4 an important target for antibody-based detection in multiple research contexts including developmental biology, neuromuscular disorders, and cancer immunology.

What are the key specifications of the DUX4 antibody with FITC conjugation?

The FITC-conjugated DUX4 antibody (ABIN2482297) is a monoclonal antibody produced in mouse that specifically targets the C-terminal region of human DUX4 protein. This antibody has been protein G purified and demonstrates specificity for a ~45 kDa protein with no cross-reactivity with DUX4c, a closely related protein . The clone identifier is P2B1 and it belongs to the IgG1 isotype. The immunogen used for antibody production consists of the C-terminal 76 amino acids of DUX4 fused with a glutathione-s-transferase (GST) tag . This antibody shows reactivity primarily with human DUX4, though cross-reactivity with mouse has also been reported. The FITC conjugation enables direct fluorescence detection without requiring secondary antibodies, making it particularly valuable for immunofluorescence applications where multicolor labeling is desired.

What experimental applications are validated for the FITC-conjugated DUX4 antibody?

The FITC-conjugated DUX4 antibody has been validated for multiple experimental applications including:

• Western Blotting (WB) - For detection of denatured DUX4 protein, typically appearing at approximately 45 kDa

• Immunohistochemistry (IHC) - For localization of DUX4 in tissue sections

• Immunofluorescence (IF) - For visualization of DUX4 in cells and tissues utilizing the FITC fluorophore

• Immunocytochemistry (ICC) - For cellular localization studies in cultured cells

The versatility across these applications makes this antibody a valuable tool for researchers investigating DUX4 expression patterns, protein interactions, and subcellular localization in various experimental contexts.

How can researchers optimize immunofluorescence protocols with FITC-conjugated DUX4 antibody for co-localization studies?

For successful co-localization studies involving the FITC-conjugated DUX4 antibody, researchers should implement the following methodological approaches:

• Spectral considerations: FITC has excitation/emission peaks at approximately 495/519 nm. When designing multi-color experiments, select compatible fluorophores with minimal spectral overlap (e.g., TRITC, Cy5) for other targets. Consider using sequential scanning if available on your confocal microscope.

• Fixation optimization: For DUX4 detection, 4% paraformaldehyde fixation for 15-20 minutes at room temperature typically preserves both antigenicity and fluorescence. Compare with methanol fixation if nuclear epitopes are being masked.

• Nuclear localization protocol: Since DUX4 functions primarily in the nucleus and interacts with nuclear proteins like STAT1, include a permeabilization step (0.2% Triton X-100 for 10 minutes) to ensure antibody access to nuclear epitopes.

• Blocking protocol: Use 5-10% normal serum from the same species as the secondary antibody (if using other non-conjugated primaries) with 0.1% BSA to minimize background fluorescence.

• STAT1 co-localization: When investigating DUX4-STAT1 interactions, the proximity ligation assay (PLA) has been successfully employed to detect close interactions between DUX4-CTD and endogenous phosphorylated STAT1-Y701 in cell nuclei , suggesting this methodology could be adapted for the FITC-conjugated antibody with appropriate controls.

• Photobleaching mitigation: FITC is relatively susceptible to photobleaching. Include anti-fade mounting media containing DABCO or propyl gallate and minimize exposure to excitation light during imaging.

What controls and validation steps are critical when using DUX4 antibody for studying FSHD muscle samples?

When employing the DUX4 antibody with FITC conjugation for FSHD research, implement these essential controls and validation approaches:

• Positive control validation: Include known DUX4-expressing cells such as transfected cells overexpressing DUX4 or FSHD patient-derived myotubes with documented DUX4 expression.

• Negative controls:

  • Technical: Primary antibody omission to assess secondary antibody non-specific binding

  • Biological: Control muscle biopsies from non-FSHD individuals

  • Competitive inhibition: Pre-incubation of antibody with immunizing peptide

• Isotype control: Include a mouse IgG1-FITC conjugated control antibody at matching concentration to evaluate non-specific binding.

• Cross-validation approaches:

  • Confirm protein expression with unconjugated DUX4 antibody (ABIN863109)

  • Parallel RNA detection methods (RT-qPCR or RNA-FISH for DUX4 mRNA)

  • Western blot validation of expected molecular weight (~45 kDa)

• Specificity testing: Verify specificity by demonstrating lack of cross-reactivity with DUX4c, as indicated in the antibody specifications .

• Expression pattern evaluation: Confirm that staining patterns match expected sporadic nuclear expression characteristic of FSHD muscle, rather than uniform staining which may indicate non-specificity.

• Functional correlation: Correlate DUX4 detection with downstream transcriptional targets known to be upregulated in FSHD muscle.

How can researchers integrate DUX4 antibody data with STAT1 interaction studies?

Research investigating DUX4-STAT1 interactions can be enhanced through strategic integration of the FITC-conjugated DUX4 antibody with complementary methodologies:

• Co-immunoprecipitation approach: Based on published findings that DUX4 interacts with STAT1, researchers can design sequential co-IP experiments where:

  • Primary IP uses anti-DUX4 to pull down protein complexes

  • Western blotting with anti-STAT1 confirms interaction

  • Reverse co-IP (STAT1 pull-down, DUX4 detection) validates bidirectional interaction

• Phosphorylation-dependent interaction analysis: Evidence indicates that phosphorylation of STAT1 at Y701 enhances interaction with DUX4 . Researchers should incorporate:

  • IFNγ stimulation conditions to induce STAT1 phosphorylation

  • Phospho-specific antibodies to detect activated STAT1

  • Comparison of interaction strength between basal and stimulated conditions

• Fluorescence microscopy co-localization workflow:

  • Use FITC-conjugated DUX4 antibody alongside a spectrally distinct STAT1 antibody

  • Apply digital image analysis with colocalization coefficients (Pearson's, Manders')

  • Quantify nuclear vs. cytoplasmic distribution of both proteins

• Proximity Ligation Assay (PLA) adaptation: Building on successful PLA application for detecting DUX4-CTD and pSTAT1-Y701 interaction , researchers can:

  • Modify protocols to work with FITC-conjugated antibody

  • Quantify interaction signals per nucleus under various stimulation conditions

  • Compare wild-type vs. mutant DUX4 interaction frequencies

• ChIP-seq integration: Combine chromatin immunoprecipitation data for both DUX4 and STAT1 to identify:

  • Genomic regions where both proteins co-occupy

  • Displacement patterns of STAT1 by DUX4 at interferon-stimulated gene promoters

  • Correlation with transcriptional suppression

What are common issues when using FITC-conjugated DUX4 antibody and their solutions?

How should researchers interpret DUX4 antibody signals in the context of interferon signaling studies?

When interpreting results from experiments using FITC-conjugated DUX4 antibody in interferon signaling contexts, researchers should consider the following methodological framework:

• Signal interpretation baseline: DUX4 has been shown to suppress IFNγ-stimulated gene expression through interaction with STAT1 . When examining DUX4 and interferon signaling:

  • Establish baseline expression of DUX4 in your cellular system

  • Document nuclear localization patterns before and after IFNγ stimulation

  • Quantify signal intensity changes following cytokine treatment

• Double-labeling interpretation guidelines:

  • Co-staining for DUX4 (FITC) and phosphorylated STAT1 requires careful signal discrimination

  • Overlap may indicate interaction but must be distinguished from coincidental colocalization

  • Quantitative approaches like intensity correlation analysis provide objective assessment

• Temporal considerations:

  • DUX4-STAT1 interactions may be dynamic following IFNγ stimulation

  • Design time-course experiments (15, 30, 60 minutes post-stimulation)

  • Record FITC signal intensity and localization changes over time

• Functional correlation approach:

  • Correlate DUX4 staining patterns with suppression of IFNγ-stimulated genes

  • Perform parallel RT-qPCR for ISGs in cells sorted based on DUX4-FITC intensity

  • Establish threshold levels of DUX4 expression needed for ISG suppression

• Comparative analysis framework:

  • Use L-L motif mutants as controls (DUX4 with mutations in conserved (L)LxxL(L) motifs)

  • Compare nuclear DUX4-FITC signal between wild-type and mutant expressing cells

  • Correlate with functional readouts of interferon response

What are advanced approaches for quantitative analysis of DUX4-FITC antibody signals?

Researchers seeking rigorous quantitative analysis of DUX4-FITC signals should implement these advanced methodological approaches:

• Advanced fluorescence intensity quantification:

  • Adopt nuclear mask-based quantification using DAPI counterstain

  • Calculate nuclear:cytoplasmic signal ratio for subcellular distribution analysis

  • Implement integrated density measurements (area × mean intensity) rather than simple intensity

• Single-cell analysis pipeline:

  • Combine FITC-conjugated DUX4 antibody with flow cytometry for population analysis

  • Establish gating strategies based on positive controls and isotype controls

  • Correlate DUX4 expression levels with phenotypic readouts at single-cell resolution

• Super-resolution microscopy approaches:

  • Apply structured illumination microscopy (SIM) or stimulated emission depletion (STED) for detailed nuclear localization

  • Quantify co-localization with STAT1 at sub-diffraction resolution

  • Map DUX4 distribution relative to chromatin organization markers

• Dynamic protein interaction assessment:

  • Implement fluorescence recovery after photobleaching (FRAP) for FITC-labeled DUX4 mobility studies

  • Quantify diffusion coefficients and immobile fractions

  • Compare dynamics between wild-type DUX4 and functional mutants

• Machine learning-based analysis:

  • Train convolutional neural networks to recognize authentic DUX4 nuclear staining patterns

  • Develop automated quantification pipelines for high-content screening

  • Implement unsupervised clustering to identify novel DUX4 distribution patterns

How does DUX4 antibody staining pattern inform understanding of FSHD pathogenesis?

The pattern of DUX4 staining using FITC-conjugated antibodies provides critical insights into FSHD pathogenesis through several key observations:

• Sporadic nuclear expression pattern: In FSHD muscle samples, DUX4 is typically expressed in only a small fraction (approximately 0.1-1%) of myonuclei. This sporadic expression pattern explains the apparent paradox of how a small number of DUX4-expressing nuclei can trigger widespread muscle pathology. Quantifying this pattern using FITC-conjugated antibodies allows researchers to correlate disease severity with the frequency of DUX4-positive nuclei.

• Propagation of pathology: Careful co-immunofluorescence studies combining DUX4-FITC with markers of cell stress reveal that nuclei adjacent to DUX4-positive nuclei often show secondary pathological changes despite lacking DUX4 expression themselves. This supports the "spreading of toxic effects" model in FSHD pathogenesis where DUX4-expressing cells affect neighboring cells through paracrine mechanisms.

• Differentiation-dependent expression: When analyzing myoblast differentiation time courses, DUX4 antibody staining demonstrates increased expression during myotube formation. This temporal pattern aligns with clinical observations that FSHD manifests progressively with muscle maturation and usage, providing insight into disease progression mechanisms.

• Correlation with chromatin structure: Combining DUX4-FITC staining with markers of chromatin organization reveals association between DUX4 expression and altered D4Z4 repeat chromatin structure, the primary genetic lesion in FSHD. This reinforces the epigenetic dysregulation model of pathogenesis.

• STAT1 signaling disruption: The demonstrated interaction between DUX4 and STAT1 visualized using co-immunofluorescence approaches helps explain immune dysregulation observed in FSHD muscle, potentially connecting DUX4 expression to inflammatory aspects of the disease.

What is the significance of DUX4 antibody detection in cancer immunology research?

The application of FITC-conjugated DUX4 antibody in cancer immunology research reveals important mechanisms of immune evasion and has therapeutic implications:

• Immune checkpoint mechanism identification: DUX4 expression in cancers suppresses IFNγ induction of major histocompatibility complex class I (MHC class I), contributing to immune evasion . Detection of DUX4 in tumor samples using FITC-conjugated antibodies allows researchers to identify this novel immune checkpoint mechanism distinct from established pathways like PD-1/PD-L1.

• Correlation with immunotherapy resistance: Quantitative analysis of DUX4 expression using immunofluorescence techniques can be correlated with response rates to immunotherapies, particularly immune checkpoint inhibitors. Higher DUX4 expression levels detected via FITC-conjugated antibodies may predict resistance to treatments that depend on intact interferon signaling.

• CIC-DUX4 fusion detection: In certain sarcomas characterized by CIC-DUX4 fusion, the fusion protein retains the C-terminal domain (CTD) of DUX4 and can inhibit IFNγ induction of interferon-stimulated genes . FITC-conjugated antibodies targeting this preserved domain enable specific detection of the oncogenic fusion protein, aiding in diagnosis and therapeutic targeting.

• STAT1 pathway modulation: The documented interaction between DUX4 and STAT1 provides a mechanistic explanation for how DUX4-expressing cancers can suppress interferon-mediated immune surveillance. Co-localization studies using FITC-DUX4 antibodies with STAT1 markers reveal potential intervention points for restoring immune recognition.

• Therapeutic vulnerability identification: By identifying tumors with high DUX4 expression using antibody detection methods, researchers can select appropriate patient populations for combination therapies targeting both DUX4-mediated immune suppression and conventional immune checkpoint pathways.

How do DUX4 antibody findings connect to evolutionary and developmental biology?

The application of FITC-conjugated DUX4 antibody in developmental and evolutionary studies provides insights into conserved biological functions:

• Evolutionary conservation analysis: Research indicates that mouse Dux, similar to human DUX4, interacts with STAT1 and suppresses IFNγ induction of interferon-stimulated genes . Comparative immunofluorescence studies using DUX4 antibodies across species help elucidate how this immune-modulatory function evolved in the DUXC family of transcription factors, potentially representing an ancient mechanism for balancing immune activation during early development.

• Zygotic genome activation (ZGA) characterization: DUX4 activates the first wave of zygotic gene expression in early embryos . Immunofluorescence studies using FITC-conjugated DUX4 antibodies in developmental models allow precise temporal and spatial mapping of DUX4 expression during this critical developmental window, connecting protein localization with transcriptional activity.

• Developmental immune privilege mechanisms: The ability of DUX4 to suppress interferon signaling may represent an evolved mechanism to create a form of immune privilege during early embryonic development. Careful immunofluorescence studies with FITC-DUX4 antibodies in embryonic tissues can test this hypothesis by correlating DUX4 expression with localized suppression of interferon response genes.

• Stem cell maintenance pathways: Detection of DUX4 in various stem cell populations using FITC-conjugated antibodies helps elucidate whether interferon suppression via STAT1 interaction contributes to stem cell niche maintenance, potentially explaining why this function has been conserved evolutionarily.

• Developmental to pathological transition: Comparative studies of DUX4 expression patterns between embryonic contexts (where it is normal) and adult tissues (where it is pathological in FSHD) provide insight into how developmental programs can be co-opted in disease states, a fundamental question in evolutionary medicine.

What emerging technologies might enhance DUX4 antibody applications in research?

Several cutting-edge technological approaches promise to expand the utility of FITC-conjugated DUX4 antibodies in research settings:

• CRISPR-mediated endogenous tagging: Future research may utilize CRISPR/Cas9 knock-in strategies to tag endogenous DUX4 with fluorescent proteins, allowing validation of antibody specificity against known fluorescent signals and enabling live-cell tracking of endogenous DUX4 dynamics in parallel with fixed-cell antibody detection.

• Spatial transcriptomics integration: Combining FITC-conjugated DUX4 antibody immunofluorescence with spatial transcriptomics techniques will allow researchers to correlate DUX4 protein localization with transcriptional changes at single-cell resolution, particularly important given the sporadic expression pattern in FSHD muscle.

• Antibody engineering approaches: Development of nanobodies or single-chain variable fragments (scFvs) against DUX4 conjugated with advanced fluorophores may overcome current limitations in tissue penetration and signal-to-noise ratio, particularly valuable for thick tissue sections or whole-mount preparations.

• Expansion microscopy compatibility: Adapting FITC-conjugated DUX4 antibody protocols for use with expansion microscopy would enable super-resolution imaging of DUX4 nuclear localization relative to chromatin organization without requiring specialized microscopy equipment.

• Mass cytometry adaptation: Conjugating DUX4 antibodies with mass cytometry tags rather than fluorophores would allow simultaneous detection of DUX4 alongside dozens of other proteins in the same cells, enabling comprehensive characterization of DUX4-expressing cells within heterogeneous populations.

• Microfluidic single-cell analysis: Integration of FITC-conjugated DUX4 antibody staining with microfluidic single-cell isolation and analysis platforms would enable correlation of DUX4 expression levels with other molecular features at unprecedented resolution.

How might DUX4 antibody research contribute to therapeutic development for FSHD?

Research utilizing FITC-conjugated DUX4 antibodies has significant potential to advance therapeutic development for FSHD through multiple avenues:

• High-throughput screening platforms: Developing cell-based assays using FITC-conjugated DUX4 antibodies enables screening of compound libraries for molecules that reduce DUX4 expression or alter its nuclear localization. Quantitative image analysis of DUX4-FITC signal provides an objective readout for therapeutic efficacy.

• Target engagement verification: For antisense oligonucleotides or siRNA approaches targeting DUX4 mRNA, FITC-conjugated DUX4 antibodies provide critical verification of target engagement by confirming reduced protein levels in muscle nuclei following treatment.

• Combinatorial therapy assessment: The documented interaction between DUX4 and STAT1 suggests potential value in combining DUX4-suppressing therapies with immunomodulatory approaches. FITC-conjugated antibodies enable assessment of how these combination therapies affect both DUX4 levels and downstream pathways.

• Biomarker development: Quantifiable parameters from DUX4-FITC immunofluorescence (percentage of positive nuclei, signal intensity distribution) could serve as biomarkers for patient stratification and treatment monitoring in clinical trials, addressing a critical need in FSHD therapeutic development.

• In vivo delivery validation: For gene therapy approaches, FITC-conjugated DUX4 antibodies applied to muscle biopsies post-treatment provide critical validation of successful in vivo delivery and functional effect of therapeutic constructs.

• Patient-derived organoid screening: Application of FITC-conjugated DUX4 antibodies to patient-derived muscle organoids allows personalized screening of therapeutic candidates, potentially identifying patient-specific response patterns.