FXYD5 Antibody

What is FXYD5 and why is it important in immunological research?

FXYD5 (FXYD Domain Containing Ion Transport Regulator 5) functions as a tissue-specific regulator of the Na,K-ATPase. It plays a critical role in inflammatory responses, particularly in the lung epithelium. FXYD5 is essential for NF-κB-stimulated epithelial production of chemokines like CCL2, which regulate immune cell recruitment to sites of inflammation . When studying inflammatory processes, particularly those involving epithelial barriers, FXYD5 antibodies allow researchers to track expression changes and localization patterns that correlate with disease progression or inflammatory responses . The protein is especially relevant for research involving barrier function, as FXYD5 overexpression has been shown to impair the alveolo-epithelial barrier, making it a key target in respiratory disease research .

What molecular forms of FXYD5 can be detected using antibodies?

FXYD5 can be detected in multiple molecular weight forms, which is important to consider when selecting antibodies and interpreting experimental results. In plasma membrane fractions of alveolar epithelial cells, FXYD5 is primarily detected as a heavily glycosylated 60-70 kDa band . In mouse lung tissue lysates, FXYD5 appears as both a major 60-70 kDa band and a minor 25 kDa band . The higher molecular weight form corresponds to the mature, heavily glycosylated FXYD5 residing at the plasma membrane, while the lower molecular weight form likely represents unglycosylated or minimally glycosylated intracellular protein . When designing experiments, researchers should consider which form they intend to detect and select antibodies that recognize epitopes preserved in the relevant form.

What are the recommended applications for FXYD5 antibodies?

FXYD5 antibodies can be utilized in multiple experimental approaches. Commonly validated applications include Western blotting (WB) for protein expression quantification, immunohistochemistry on paraffin-embedded sections (IHC-P) for tissue localization studies, and enzyme immunoassays (EIA) for protein detection in solution . For Western blotting, conditions should be optimized to detect the appropriate molecular weight forms (either 60-70 kDa glycosylated or 25 kDa non-glycosylated forms) . For immunohistochemistry, antibodies targeting the middle region (e.g., amino acids 70-100) have been validated and can effectively localize FXYD5 in tissue sections . The experimental design should carefully consider the specific epitope recognition of the antibody, particularly when studying functional domains or post-translational modifications of FXYD5.

How can researchers distinguish between different glycosylation states of FXYD5 in experimental samples?

Distinguishing between glycosylation states of FXYD5 requires specific methodological approaches. Researchers can employ enzymatic deglycosylation using peptide N-glycosidase F (PNGase F) for N-linked glycans or O-glycosidase for O-linked glycans, followed by Western blotting to observe mobility shifts . Since FXYD5 is heavily O-glycosylated at the plasma membrane (appearing as a 60-70 kDa band), while intracellular forms may be unglycosylated (appearing as a 25 kDa band), this approach allows researchers to determine the subcellular fraction and modification state of the protein . Additionally, subcellular fractionation combined with surface biotinylation can be used to isolate plasma membrane-associated FXYD5 specifically before antibody detection . These techniques are particularly valuable when investigating how glycosylation status affects FXYD5 function in inflammatory signaling or when evaluating translocation of FXYD5 to the plasma membrane in response to stimuli like LPS.

What are the critical controls needed when using FXYD5 antibodies in inflammation models?

When investigating FXYD5's role in inflammation, several critical controls must be implemented. First, researchers should include FXYD5 silencing controls (via siRNA or shRNA) to confirm antibody specificity and establish baseline expression levels . The search results indicate that different silencing approaches can produce up to 70% reduction in FXYD5 expression, which should be reflected in antibody-based detection methods . Second, time course experiments are essential, as FXYD5 shows dynamic regulation in response to inflammatory stimuli like LPS (with peak mRNA expression occurring around 6 hours post-stimulation) . Third, when studying membrane localization, researchers should compare total lysate preparations with surface biotinylated fractions to distinguish between total protein expression and membrane-localized protein . Finally, when analyzing downstream inflammatory markers (such as CCL2, IL-6, or TNF-α), appropriate positive controls (cytokine standards) and negative controls (vehicle-treated samples) should be included to validate the relationship between FXYD5 expression and inflammatory response .

How do FXYD5 expression patterns differ between normal and inflamed tissues, and how should antibody-based detection be optimized accordingly?

• Use antibodies that recognize epitopes preserved across different glycosylation states

• Implement subcellular fractionation to distinguish membrane versus intracellular pools

• Utilize time-course experiments to capture dynamic expression changes (peaks observed at 6-24 hours post-stimulation in LPS models)

• Consider dual-labeling approaches that simultaneously detect FXYD5 and inflammatory markers like phosphorylated IκBα

The shift in glycosylation status is functionally significant, as the glycosylated plasma membrane form appears to play a direct role in mediating inflammatory responses through NF-κB pathway activation .

What is the optimal protocol for using FXYD5 antibodies in Western blotting of lung tissue samples?

For optimal Western blotting of FXYD5 in lung tissue samples, the following protocol is recommended based on the research findings:

• Sample preparation:

  • Homogenize lung tissue in RIPA buffer with protease and phosphatase inhibitors

  • Centrifuge at 14,000g for 15 minutes at 4°C to remove debris

  • For plasma membrane isolation, perform surface biotinylation followed by streptavidin pull-down

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation of both high (60-70 kDa) and low (25 kDa) molecular weight forms

  • Load 20-40 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF membranes at 100V for 1 hour or 30V overnight at 4°C

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Use antibodies targeting the middle region (AA 70-100) of FXYD5 at 1:1000 dilution

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST (5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour

• Detection:

  • Develop using enhanced chemiluminescence

  • When analyzing results, note that FXYD5 appears as both a major band at 60-70 kDa (glycosylated form) and a minor band at 25 kDa (non-glycosylated form) in lung tissue lysates

This protocol should allow researchers to effectively detect both glycosylated and non-glycosylated forms of FXYD5 in lung tissue samples.

How can researchers effectively use FXYD5 antibodies to study NF-κB pathway activation in epithelial cells?

To effectively study NF-κB pathway activation using FXYD5 antibodies, researchers should implement a multi-parameter analytical approach:

• Dual immunostaining technique:

  • Use FXYD5 antibodies (targeting AA 70-100) together with antibodies against phosphorylated IκBα to monitor correlation between FXYD5 expression and NF-κB activation

  • Include time-course analysis (5min, 15min, 30min, 1h, 2h) to capture dynamic relationships between FXYD5 localization and IκBα phosphorylation/degradation

• FXYD5 silencing experiments:

  • Implement siRNA or shRNA knockdown of FXYD5 (aim for ≥70% reduction)

  • Assess effects on both basal and stimulated NF-κB activation by measuring:
    a) IκBα phosphorylation by Western blot
    b) Nuclear translocation of p65 by immunofluorescence
    c) NF-κB-dependent gene expression by qRT-PCR

• Pathway validation:

  • Use specific stimuli targeting different receptors (LPS for TLR4, TNF-α for TNFR, IFN-α for IFNAR)

  • Monitor whether FXYD5 is required for NF-κB activation across all pathways or specific to certain stimuli

  • Research has shown that FXYD5 silencing prevents NF-κB activation downstream of multiple cytokine receptors, not just TLR4

• Functional readouts:

  • Measure secretion of CCL2, IL-6, and TNF-α as functional consequences of FXYD5-mediated NF-κB activation

  • Correlate cytokine production with FXYD5 expression levels and membrane localization

This comprehensive approach allows researchers to establish the mechanistic relationship between FXYD5 expression/localization and NF-κB signaling in epithelial cells.

What techniques can be used to validate FXYD5 antibody specificity in experimental systems?

Validating FXYD5 antibody specificity is crucial for experimental rigor. The following techniques are recommended based on research practices:

• Genetic knockdown validation:

  • Implement shRNA or siRNA targeting FXYD5 (targeting 70% reduction in expression)

  • Compare antibody signal between knockdown and control samples by Western blot, IHC, and flow cytometry

  • A specific antibody should show proportional reduction in signal intensity corresponding to knockdown efficiency

• Overexpression validation:

  • Express tagged FXYD5 constructs (e.g., mCherry-HA-FXYD5) in cell lines

  • Confirm co-localization of antibody signal with tag-specific antibodies or fluorescent protein signal

  • This approach is particularly valuable for validating subcellular localization patterns

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide (for the AA 70-100 region) before application to samples

  • A specific antibody signal should be significantly reduced or eliminated

  • This technique is especially relevant for antibodies generated using peptide immunogens like the one described in search result

• Cross-platform validation:

  • Compare protein detection patterns across multiple techniques (WB, IHC, flow cytometry)

  • Verify that molecular weight, expression patterns, and subcellular localization are consistent across platforms

  • For FXYD5, confirm detection of both 60-70 kDa (glycosylated) and 25 kDa (non-glycosylated) forms where appropriate

• Tissue-specific expression:

  • Compare antibody staining patterns with known tissue expression profiles

  • FXYD5 is abundantly expressed in ATII cells in the lung, providing a reference point for validation

These validation approaches ensure that experimental findings attributed to FXYD5 are genuinely reflecting the biology of this protein rather than non-specific antibody interactions.

How should researchers interpret changes in FXYD5 expression patterns during inflammatory responses?

When interpreting changes in FXYD5 expression during inflammation, researchers should consider several key parameters:

• Temporal dynamics:

  • FXYD5 mRNA expression peaks approximately 6 hours after LPS stimulation in lung tissue

  • Protein expression changes may lag behind mRNA changes, with significant increases observed between 4-24 hours post-stimulation

  • These temporal patterns provide important context for interpreting expression data at single time points

• Molecular weight shifts:

  • An increase in the ratio of 60-70 kDa to 25 kDa forms indicates enhanced post-translational modification and membrane trafficking

  • This shift correlates with increased inflammatory signaling capacity

  • Changes in glycosylation patterns may precede changes in total FXYD5 protein levels

• Localization changes:

  • Increased plasma membrane localization (detectable via surface biotinylation) is functionally significant even if total protein levels show modest changes

  • Subcellular redistribution from intracellular compartments to the plasma membrane corresponds with enhanced inflammatory signaling

• Correlation with inflammatory markers:

  • FXYD5 expression changes should be analyzed alongside downstream markers like phosphorylated IκBα, CCL2 production, and immune cell recruitment

  • The research demonstrates that FXYD5 levels correlate with these inflammatory parameters

• Cell-type specificity:

  • Changes in FXYD5 expression in alveolar epithelial cells have different functional implications than changes in other cell types

  • Cell-type specific analysis provides greater mechanistic insight than whole-tissue measurements

Understanding these interpretative frameworks helps researchers distinguish between correlation and causation when analyzing FXYD5 expression data in inflammatory contexts.

What are common technical challenges when using FXYD5 antibodies and how can they be addressed?

Researchers may encounter several technical challenges when working with FXYD5 antibodies:

• Glycosylation heterogeneity:

  • Challenge: Variable glycosylation patterns can affect epitope accessibility and produce inconsistent banding patterns

  • Solution: Use deglycosylation enzymes (PNGase F, O-glycosidase) to reduce heterogeneity before Western blotting

  • Alternative: Select antibodies targeting regions less affected by glycosylation (such as the middle region AA 70-100)

• Membrane protein solubilization:

  • Challenge: As a membrane-associated protein, FXYD5 may be difficult to extract efficiently

  • Solution: Use detergent-based lysis buffers containing 1% Triton X-100 or NP-40 with brief sonication

  • For heavily glycosylated forms, include 0.1% SDS in lysis buffer while maintaining non-denaturing conditions for immunoprecipitation

• Signal specificity in tissues with low expression:

  • Challenge: Distinguishing specific signal from background in tissues with low FXYD5 expression

  • Solution: Implement FXYD5 silencing or overexpression controls in parallel

  • Use tyramide signal amplification for IHC applications to enhance sensitivity while maintaining specificity

• Cross-reactivity with other FXYD family members:

  • Challenge: The FXYD family contains several related proteins that may cross-react with some antibodies

  • Solution: Validate antibody specificity using cells overexpressing different FXYD family members

  • Select antibodies targeting unique regions of FXYD5 rather than the conserved FXYD domain

• Detection of dynamic changes in response to stimuli:

  • Challenge: Capturing rapid changes in FXYD5 expression or localization

  • Solution: Implement tight time-course experiments (e.g., 0, 15min, 30min, 1h, 2h, 4h, 6h, 24h)

  • Use subcellular fractionation to track movement between compartments rather than relying solely on total expression levels

Addressing these challenges through appropriate technical modifications enhances the reliability and reproducibility of FXYD5 antibody-based experiments.

How can FXYD5 antibodies be utilized to study immune cell recruitment in inflammatory lung models?

FXYD5 antibodies can be leveraged to investigate immune cell recruitment mechanisms in inflammatory lung models through several sophisticated approaches:

• Dual-color flow cytometry:

  • Use FXYD5 antibodies in combination with immune cell markers to analyze correlations between FXYD5 expression and specific immune cell populations

  • Research has shown that FXYD5 overexpression specifically enhances recruitment of interstitial macrophages (CD11b^hi MHCII^hi), classical monocytes (CD11b^hi MHCII^low Ly6C^hi), and eosinophils (SiglecF^hi CD11c^low)

  • Gating strategies should first identify myeloid cells using CD45, then further characterize subpopulations using specific markers

• Conditional modulation systems:

  • Implement cell-type specific FXYD5 modulation (e.g., epithelial-specific overexpression using Ad-FXYD5)

  • Track immune cell recruitment in these models using flow cytometry and immunofluorescence

  • Correlate with cytokine/chemokine production (especially CCL2, which recruits CCR2+ monocytes)

• Mechanistic dissection:

  • Use CCR2 neutralizing antibodies or CCR2-knockout mice to determine the specificity of FXYD5-mediated immune cell recruitment

  • Research has demonstrated that FXYD5-induced cell recruitment is decreased by blocking CCR2 signaling, confirming a mechanistic link between FXYD5, CCL2 production, and immune cell recruitment

• Temporal relationship analysis:

  • Implement time-course studies tracking FXYD5 expression, cytokine production, and immune cell recruitment

  • FXYD5 expression changes precede cytokine production, which in turn precedes immune cell recruitment, establishing causality

These approaches allow researchers to establish not just correlative but causal relationships between FXYD5 expression and specific patterns of immune cell recruitment in inflammatory lung disorders.

What are the considerations for using FXYD5 antibodies in multiplexed imaging systems?

When incorporating FXYD5 antibodies into multiplexed imaging systems, researchers should consider several technical and biological factors:

• Epitope preservation in multiplexed protocols:

  • Challenge: Some multiplexing techniques (especially those involving sequential stripping/reprobing) may compromise epitope integrity

  • Solution: Position FXYD5 detection early in sequential protocols or use spectral unmixing approaches with simultaneous antibody application

  • For heavily modified forms of FXYD5, validate epitope stability after fixation and any antigen retrieval steps

• Cross-talk with other channels:

  • Validate signal specificity in multiplex settings by comparing with single-staining controls

  • Select fluorophores with minimal spectral overlap for FXYD5 and co-markers (e.g., NF-κB pathway components, cellular junction proteins)

  • Consider using primary antibodies from different host species to minimize secondary antibody cross-reactivity

• Biological co-localization analysis:

  • Design multiplexed panels to simultaneously assess:
    a) FXYD5 expression and subcellular localization
    b) Cell junction proteins (FXYD5 disrupts epithelial junctions)
    c) Inflammatory signaling components (phospho-IκBα, nuclear p65)
    d) Cell-type markers to distinguish epithelial cells from infiltrating immune cells

• Quantitative considerations:

  • Implement appropriate controls for potential autofluorescence (particularly in lung tissue)

  • Establish consistent thresholding parameters for co-localization analysis

  • Use computerized image analysis to quantify co-expression patterns across multiple experimental conditions

• 3D tissue analysis:

  • For studying barrier function, consider using thick tissue sections (50-100 μm) with confocal or light-sheet microscopy

  • FXYD5 can be detected alongside tight junction proteins to assess barrier integrity in three dimensions

  • Computational analysis can quantify spatial relationships between FXYD5 expression and barrier disruption

These considerations optimize the use of FXYD5 antibodies in advanced imaging applications, enabling researchers to visualize the molecular mechanisms connecting FXYD5 expression to inflammatory signaling and barrier dysfunction.